Anthelmintic resistance—The state of play

Br. vet.J. (1993). 149, 123

REVIEW A N T H E L M I N T I C R E S I S T A N C E - - T H E STATE O F PLAY

F.JACKSON Moredun Research Institute, Edinburgh

SUMMARY There is evidence that the incidence of anthelmintic resistance is increasing in livestock in countries throughout the world including the United Kingdom. Early detection of emerging drug resistance is important since reversion to susceptibility appears not to occur in highly selected homozygous strains. Because the current in vivo and in vitro assays, which generally determine the degree of disruption of normal physiological function of different parasite stages, are relatively insensitive, effort is being made to develop more direct genetic and biochemical diagnostic assays. Studies on the selection and genetics of resistance suggest that resistance is normally polygenic and arises from within the normal phenotypic range and that there are three phases in the selection process. An initial susceptible phase is followed by an intermediate one in which heterozygous resistant individuals are common within the population and finally homozygous resistant individuals predominate within the population. For these reasons low efficacy treatments, which enable the survival of heterozygous resistant individuals, and suppressive regimes, which only allow homozygous resistant individuals to survive, increase the rate of development of drug resistance. Strategies to delay the onset of resistance and control resistant strains usually incorporate minimal chemoprophylaxis, seek to maximize drug efficacy, and if possible include a 'slow' drug rotation and seek to limit host parasite contact by manipulation of the grazing environment. Although multi-species mathematical models of anthelmintic resistance appear to offer a means of assessing the long term impact of these and other control strategies, current models are limited by a lack of detailed biological knowledge. In particular, more information on the status and numbers of alleles associated with resistance to specific drugs, their frequencies within populations of different species and the fitness of resistant and susceptible populations is required. /Mathelmintic resistance provides an example of the adaptability of metazoan parasites under intensive selection and suggests that sustainable control strategies will require an integrated approach in which both 0007/1935/93/020123-16/$08.00/0

© 1993 Bailli~re Tindall

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BRITISH VETERINARYJOURNAL; 149, 2 c h e m o t h e r a p y and i m m u n o t h e r a p y , together with environmental mana g e m e n t are used to control nematodoses.

INTRODUCTION

Resistance o f parasites to c h e m o t h e r a p e u t i c agents has b e c o m e an increasingly widespread p r o b l e m in r e c e n t years and is now a cause for c o n c e r n for all those interested in controlling protozoal, helmilath and a r t h r o p o d parasites of veterinary and medical importance. T h e e m e r g e n c e o f resistant strains of parasites has led to research focused not only u p o n the aetiology, diagnosis and control of resistance but has also provided the spur for research into alternative means of parasite control. In particular, the advances currently being m a d e into the develo p m e n t o f anti-parasite vaccines and selection o f b r e e d lines displaying an increased imnaunological responsiveness to gastrointestinal n e m a t o d e s are in part attributable to the increased levels of parasite resistance that have been seen t h r o u g h o u t the world. Since it may be many years before such alternative means of controlling h e l m i n t h infections b e c o m e widely available, c h e m o t h e r a p y will necessarily remain as an i m p o r t a n t means o f achieving control. T h e o t h e r advantages offered by chenaotherapy, particularly those associated with animal weltare, suggest that there will always be n e e d fi)r anti-parasite drugs (Gutteridge, 1989). For these reasons it is i m p o r t a n t that the efficacy of currently established c h e m o therapeutic agents and any novel drugs that are d e v e l o p e d in future is conserved. In the UK, the immediate c o n c e r n must be to conse~-ve the efficacy o f broad spectrum drugs (ie drugs within the benzimidazole, imidazothiazole and avermectin families) which have different modes of action and are currently most often used to control nematodoses. Altlaough increasing levels o f drug resistance may a p p e a r to be an inexorable process as a c o n s e q u e n c e of routine d r u g treatment, an integrated a p p r o a c h utilizing the findings fi'om recent studies on the aetiolo~, and p r e v e n t i o n of resistance c o u p l e d with the newer technologies o f m o l e c u l a r genetics and m a t h e m a t ical modelling offer the best means o f minimizing the impact of anthelmintic resistance.

D E T E C T I N G RESISTANCE

Anthelnfintic resistance is d e f i n e d ill terms of d r u g effectiveness against a population of nematodes. Resistance is c o n s i d e r e d to have been selected when a previously effective d r u g ceases to be so, due to selected heritable changes in the exposed population. T h e in vivo and in vitro m e t h o d s that may be used to detect resistance are stmHnal-ized in Table I. All of the m e t h o d s have drawbacks e i t h e r in terms of cost, applicability, and i n t e r p r e t a t i o n / r e p r o d u c i b i l i t y of findings. T h e most widely used tests to verit\, resistance are the in vivo c o n t r o l l e d efficacy test and f:aecal egg c o u n t reduction test which have a wide applicability since they are used also in the selection o f novel c o m p o u n d s . Despite being costly and having a p o o r ability to detect low levels of resistance (Martin el aL, 1989) their universal applicabiliD' ensures that these assays will c o n t i n u e to provide the yardstick against

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which all o t h e r tests will be m e a s u r e d . Most o f the m e t h o d s listed in T a b l e I are bioassays which d e t e r m i n e the d e g r e e o f d i s r u p t i o n o f n o r m a l physiological function o f parasites e x p o s e d to the test c o m p o u n d . Given an i m p r o v e d u n d e r s t a n d ing o f b o t h the m o d e of action o f drugs a n d the m e c h a n i s m s o f d r u g resistance, t h e n b i o c h e m i c a l a n d genetic assays, which offer a m o r e direct m e a s u r e m e n t o f resistance by i d e n t i ~ i n g the g e n e s a n d or b i o c h e m i c a l processes associated with resistance, may oiler the best p r o s p e c t of d e t e c t i n g resistance at the earliest possible stage. A l t h o u g h standardization o f field (in vivo) tests such as the faecal egg c o u n t r e d u c t i o n tests theoretically offer a m e a n s of c o m p a r i n g the d e g r e e o f resistance shown by different ecotypes selected using different drugs, evidence is e m e r g i n g that such tests may n e e d to be host a n d drug-specific. P o s t - t r e a t m e n t samples in faecal egg c o u n t reductiorl tests are lmrmally taken a r o u n d 10 to 14 days after d r u g administration. However, a r e c e n t study using h o u s e d goats naturally infected with a multiple resistant strain o f Teladorsa~a (Osterlagia) a n d lambs artificallv infected with the s a m e strain has shown that inhibition o f egg o u t p u t may o c c u r over this p e r i o d as a c o n s e q u e n c e o f ivermectin t r e a t m e n t (Jackson unp u b l i s h e d data). T a b l e II shows the weekly faecal egg counts of two artificially infected lambs a n d calculated efficacy d u r i n g a 4-week p e r i o d following t r e a t m e n t with ivermectin.

PREVAI,ENCE OF ANTHELMINTIC RESISTANCE Resistance has b e e n r e c o r d e d in m a n y countries t h ' r o u g h o u t the world against drugs in all of the three b r o a d s p e c t r m n families, the benzimidazoles, a v e r m e c t i n a n d imidazothiazoles, which are c o m m o n l y used by the livestock industries to control n e m a t o d o s e s (see reviews by Prichard el aL, 1980; Coles, 1986; Waller & Prich-

Table I In vivo and in vitro bioassays (BA), biochemical assays (BC) and genetic assays (G)

used in the detection of anthelmintic resistance A vsay

S/u,ct~Tim

Controlled test Fgg count reduction Egg hatch assay ( 1) Egg hatch assay (2) l,mval paralysis ( 1) l,arval paralysis (2) l,mxal development ( I ) l.arval development (2) l.arval dewlopment (3) l.m-val development (4) Tuhulin hinding Estcrase activity Tuhulin prohc ( 1) Tubulin prohc (2)

All drugs All drugs BZ BZ LV

Assay type

In vivo BA lit vivo BA In vitro BA In vitro BA In vitro BA IV In viho BA BZ, IV In vitro BA BZ, I.V In vitro BA BZ, IV, I.V In vilro BA BZ, IV, IN In vitro BA BZ In vitro BC BZ In vitro BC BZ In vitro G BZ In vitro (;

Application

A uthor(s)

Powers et al. (1982) Widespread Presideme (1985) Widespread l.eJambre (1976) Widespread Hunt & Taylor (1989) Widespread Research Martin & LeJambre (1979) Gill et al. (1991) Research Coles & Simpkin (1977) Research Tavlo, (1990a) Research l.acey et al. (1990) Resemch Research Hubert & Kerboeuf (1992) I.acev & Snowden (1988) Resea,ch Sutherland et al. (1989) Research Roos et al. (1990) Research l.eJambre (1990) Resca,ch

BZ, bcnzimidazolc drugs; I.V, levamisole; IV, ivcrmectin.

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Table II Weekly faecal egg counts (efficacy %) of lambs infected with 10000 L~ of a caprine ivermectin resistant strain and treated with ivermectin at 2 0 0 / l g / k g bodyweight Lamb number

Day 0

Day 7

Day 14

Day 21

Day 28

1

450

0 (100%)

24 (94.6%)

126 (72%)

117 (74%)

2

256

0 (100%)

0 (100%)

153 (40.2%)

225 (12.1%)

ard, 1986; Waller, 1987; Prichard, 1990). Resistance has been recorded also in drugs with a narrower spectrum of activity such as the salicylanilides (Rolfe et aL, 1990). Many of the earliest reports of ruminant nematode strains resistant to broad spectrum anthelmintics emanated from the Southern hemisphere and usually involved species with a high biotic potential such as Haemonchus contortus and Trichostrongylus colubriformis. The rate of emergence of resistance appears to vary geographically in accordance with the prevailing climate, parasite species and treatment regimes adopted in the region. Although to date there have been relatively few reports of anthelmintic resistance in cattle nematodes (Prichard, 1990) it is not clear whether this situation results largely from host or parasite characteristics or simply reflects differences in bovine treatment regimes which may reduce parasite exposure. In the past there was a tendency for sheep and goat producers to adhere to treatment regimes involving frequent treatment and consequently, as the prevalence of resistance within one family increased, a switch to alternative families often resulted in the emergence of multiple resistance (Van Wyk & Malan, 1988; Badger & McKenna, 1990; Watson & Hosking, 1990). Although the rate of emergence of resistant strains has generally been slower in temperate zones in the northern hemisphere, the prevalence of resistance is also increasing throughout Europe (Borgsteede, 1990; Bjorn et al., 1990; Taylor, 1990b; Scott et al., 1990; Waller et al., 1990). Prevalence in the UK There can be no doubt that there has been an increase in benzimidazole resistance in small ruminants and horses in the UK in recent years (Taylor, 1990b). Surveys conducted during 1990 on sheep farms in England (Coles et al., 1991) and in Scotland (Mitchell et aL, 1991) showed prevalence rates of 53% and 24% respectively. In the former study, benzimidazole resistant nematodes were detected on 45% of the farms in Oxfordshire, 37% of farms in East Sussex and 74% of farms in West Sussex. The prevalence of benzimidazole resistance recorded by Coles and his co-workers is considerably higher than that seen in earlier studies in the region (Taylor, 1990b). Although no large scale surveys of resistance in other host species have been undertaken, limited surveys using fibre-producing goat herds in Scotland have also shown a high prevalence of benzimidazole resistance. In the first study, five of six farms screened were positive (Jackson et al., in press). Four more herds were surveyed the following season, two of which proved to be positive (Jackson, unpublished data). A small survey involving nine riding establishments

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in Scotland identified benzimidazole-resistant cyathostomes in horses on two of the properties (King et al., 1990). Confirmed reports.of resistance to other drug families in the UK are much rarer. Recently, a strain of Teladorsag~a has been isolated from hill goats in Scotland which showed some resistance to ivermectin and is also resistant to the benzimidazoles (Jackson et al., 1992).

DEVELOPMENT OF RESISTANCE

Anthelmintic resistance in nematodes is thought to be a pre-adaptive phenomenon and so for many species of nematode the gene or genes conferring resistance are available within the population prior to first exposure to a drug. Thus, anthelmintic resistance arises from selection within the normal phenotypic range, in contrast to the situation with persistent insecticides where selection favours rare mutations outside the normal phenotypic range (McKenzie, 1985). There appear to be three phases in the selection process (Prichard, 1990). Firstly, an initial phase of anthelmintic susceptibility occurs where the frequency of resistant individuals within the population is low. Given continued exposure to a drug, an intermediate phase then develops in which the frequency of heterozygous resistant individuals within the population increases. Finally, sustained selection pressure results in a resistant phase where homozygous resistant individuals predominate within the population. Since the selection of resistance is most rapid when both heterozygous and homozygous individuals survive treatment, underdosing, which enables survival of the former, can play a key role in influencing the rate of development of resistance. Low efficacy treatments administered to sheep have been shown rapidly to select resistant Teladorsagia strains (Martin, 1990). The tendency to treat goats at the same level as sheep may, because of differences in drug pharmacodynamics (Gahier et al., 1981; Bogan et al., 1987) and the degree of rumen bypass (Sangster et al., 1991), result in reduced bioavailability in the former species which is an important factor accounting for the high prevalence of anthelmintic resistance in goat herds. Studies on the efficacy of levamisole (Coles et al., 1989) and oxfendazole (Sangster et al., 1991) in goats have shown that goats require higher dose rates than sheep. The results of an epidemiological study on anthelmintic resistance in fibre-producing hill goats suggest that a reduced efficacy against inhibited L.,s (Jackson, unpublished data) played an important part in the selection of heterozygous ivermectin resistance (Jackson et al., 1992). j Although simple logic suggests that if underdosing increases the rateJof development of resistance then increased dose rates should delay the development of resistance, mafortunately there is little evidence to support the correctness of this simplistic view. Overdosing not only has obvious drawbacks in terms of tissue residues, increased toxicity with certain drugs and cost but also has been shown to offer little benefit in terms of both systemic availability and efficacy in goats (Sanger et aL, 1991). Because broad spectrum anthehnintics are normally administered at rates which ensure that only homozygous resistant individuals survive, treatment frequency also influences the rate of selection of resistance. Suppressive treatment

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regimes, in which animals are treated within or close to the prepatent period of the parasite population, inevitably favour the selection of a parasitic population (infrapopulation) which contains only resistant phenotypes. The continued use of such regimes in the New Zealand goat industry has resulted in a high incidence of benzimidazole resistance and the emergence of multiple resistance (Badger & McKenna, 1990; Watson & Hosking, 1990). The population dynamics of the free living stages (suprapopulation) are another important factor that can influence the rate of development of resistance, particularly in temperate climates. For species such as Teladorsagia spp., the size of this population in r~ugia on permanent pasture in the UK tends to follow a stable annual pattern. The prevailing moist and mild climate coupled with the relatively long survival times for infective larvae of this species generally efisures that the population does not fluctuate abruptly. The stability of these epidemiological patterns may, when a drug is first used, slow the rate of selection of resistance since initially they can provide a reservoir of susceptibility. Unfortunately, the converse is also true and highly selected resistant populations may survive for some time on pasture. For other species in the UK, such as Haemonchus, the suprapopulation may show a marked seasonal decline during the late winter/early spring and treatments administered to ewes at such times may exert a high selection pressure (Taylor, 1990b). Dose and move strategies involving 'safe', minimally contaminated grazing can similarly increase the rate of development of resistance (Taylor & Hunt, 1989; Smith, 1990). In other regions, treatments given during climatic extremes which reduce the size of the suprapopulation and hence alter the infrapopulation/suprapopulation ratio, have also been shown to increase the rate of development of anthelmintic resistance (Donald & Waller, 1982; Martin et al., 1989; Smith, 1990). The question of 'fitness' of strains undergoing selection is an important one, since preventive strategies such as those that involve the use of an annual, 'slow' rotation of drugs rely upon the possibility of reversion to susceptibility occurring in the intervening ),ears between treatments with a single drug. The earliest attempts to measure fitness of parasite strains within the laboratory provided conflicting findings and have been considered as ecotypic since different strains were used in the investigations. Studies using selected strains from the same lineage provide little evidence of any reduction in fitness of homozygous resistant individuals. They do, however, provide support for the view that if reversion is to occur then it is only likely to do so during the heterozygous-resistant phase of development, before selection pressure results in co-adaptation with general fitness characteristics (Maingi et al., 1990; Scott et al., 1991). Field studies in Australia also provide little or no evidence of reversion in the absence of drug therapy; in one study conducted over four years during which time there was no exposure to the benzimidazoles, no reduction in benzimidazole resistance was detected. Over the same period, levamisole treatment did produce some reduction in benzimidazole resistance but the level of resistance was still considered too high for the re-introduction of the benzimidazoles. Moreover, levamisole resistance also became apparent during the study (Martin, 1990). The findings from reversion studies in Europe are similar and no reversion to benzimidazole susceptibility was evident after six years of levamisole treatment in one study (Borgsteede & Duyn, 1989)

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and after nine years of alternate levamisole and ivermectin treatment in another (Jackson, unpublished data).

GENETICS OF RESISTANCE

Selection models for anthelmintic resistance where treatments are usually of short-persistence and discriminatory, having a high but not absolute efficacy, suggest that resistance develops from the upper phenotypic range of susceptibility. Resistance selected in this manner will generally fall under control of more than one gene (Martin, 1990). The number of resistance alleles, degree of dominance, fitness of resistant genes and extent of integration into the gene pool are important specific determinants influencing the rate of development of anthelmintic resistance (Georghiou & Taylor, 1977). Recent studies have progressed our understanding of the genetic mechanisms involved in resistance to anthelmintics. Mendelian studies with backcrosses (Le Jambre, 1985) and an appropriate bioassay to determine resulting level of resistance suggest that benzimidazole resistance for the common sheep nematodes such as Haemonchus, T. colubnformis and Teladorsagia is polygenic (Martin, 1990). However, the findings from simple backcross studies may be ambiguous due to overlapping concentration response lines and thus the question of monogenic against polygenic inheritance of benzimidazole resistance is still an area of interest (Roush, 1990). However, backcross studies conducted with a levamisole resistant strain of T. colubriformis suggest that resistance in this case may be due to a major sex-linked gene, single recessive gene or linked gene complex (Martin & McKenzie, 1990a). Little is known about the genetics of ivermectin resistance in parasitic nematodes but if the mechanisms are similar to those in the free living nematode Caenorhabditis elegans then ivermectin resistance may be under polygenic control (LeJambre, 1990). Molecular biological techniques offer the best methods for determining the number of genes involved in resistance and for elucidating the mechanisms of resistance. Most of the studies to date on parasitic nematodes have focused upon strains resistant to the benzimidazoles, which act as anthelmintics by binding to tubulin and limiting polymerization of at and fl subunits (Lacey & Prichard, 1986). Benzimidazole resistance in Haemonchus appears to be due to structural changes in the target protein (s) and riot to differences in benzimidazole transport or gene amplification of the target proteins (Roos & Boersema, 1990). Resistance to this drug family appears to involve the deletion of one or more susceptible/3 tubulin genes (LaJambre, 1990) and thus in one study (Roos et al., 1990) different strains of resistant Haemonchus were almost monomorphic for resistant/3 tubulin. Studies with benzimidazole resistant clones of C. elegans have shown that resistance maps to a single/3 tubulin gene, the ben-I gene which encodes a/3 tubulin sensitive to the effects of drugs within this family (Driscoll et al., 1989). In some of the resistant mutants this particular gene was absent and in others the gene was not apparently expressed. Assuming that resistance develops in the same way in parasitic nematodes, then the initial selection process might lead to populations in which inactivation of susceptible tubulins was common. Further selection would then

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result in a population in which the majority of the individuals had completely deleted the gene for susceptible tubulins (LeJambre, 1990). Although lack of knowledge concerning the mode of action of drugs and their target proteins imposes limits upon the rate of progress, a number of powerful tools exist within the molecular biological arsenal and can be used to research further into the genetics of resistance (Le Jambre, 1990). Where target proteins are unknown, random restriction fragment length polymorphisms (RFLP) a n d / o r simple sequence length polymorphisms (SSLP) can be used to identify DNA polymorphisms which are associated with the resistance gene(s). Once identified, the DNA regions associated with resistance may then be amplified using the polymerase chain reaction (PCR) and could be used to probe a genomic library to identify the genes that regulate resistance to a drug (LeJambre, 1990).

MODELS OF RESISTANCE

A better understanding of the genetics of anthelmintic resistance is one prerequisite for the development of mathematical models. Soundly constructed multi-component models offer powerful tools for investigating the impact over the long term of different control strategies. Currently, the development of multi-species models that can provide useful quantitative data is limited only by a lack of detailed biological knowledge in certain areas. In particular, models require information on the status and numbers of alleles associated with resistance to specific drugs, their frequencies within populations of different species and the fitness of resistant and susceptible populations. Despite the limitations within the current field of knowledge, existing anthelmintic resistance models have attempted to determine the impact of various control strategies upon selection for resistance. One of the first species for which a model of anthelmintic resistance was developed was Teladorsagia circumcincta (Gettinby, 1989; Gettinby et al., 1989). This network flow simulation model incorporates not only the biological aspects of the life cycle but also incorporates differences in prevalence of resistance alleles in first generation, mortality in refugia, proportion of suprapopulation to encounter host and genetic fitness of the different genotypes in single and two locus models. A single locus model, with a frequency of homozygous resistance of 0.1%, comparing dose and move (two treatments per annum) with permanent pasture (four doses per annum) shows that the entire population becomes resistant on permanent pasture after 12 years. Dose and move strategies appear to select less heavily for resistance, after 20 years 83% of the population consists of resistant genotypes. Interestingly, this model has been used also to examine the influence of changing temperature and biotic potential of the parasite. An increase in temperature alone exerts only a marginal effect upon the development of resistance but if allied to a 10% increase in fecundity and establishment can double the rate of development of resistanCe (Gettinby et al., 1990). Another specific model incorporating level of resistance, host mortality and acquired immunity has been developed for the intestinal parasite T. colubriformis (Barnes & Dobson, 1990). This model is predictive for lambing and weaning time, s h e e p / p a d d o c k rotation, different drug treatments and the use of controlled

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Table 3 Influence upon the composition of the next generation o f the proportions of the suprapopulation and post treatment infrapopulation Proportion in refugia 10% 10% 90% 90%

Efficacy of drug

Contributionto next generation

99% 90% 99% 90%

8.0% 47.0% 0.1% 1.0%

release devices. The model allows for up to three genes with two alleles giving a maximum of 27 genotypes for one drug or three genotypes for drugs within each of the three drug families. This model also suggests that grazing management can play a dominant role in parasite control. The use of controlled release devices will under certain circumstances reduce mortalities and production losses and may in some cases not cause a substantial increase in anthelmintic resistance for up to five years. Non-specific models for resistance have been described by Smith (1990) and Martin (1990). The former author has produced a general model for direct life cycle parasites in which there is some overlap of generations incorporating single host resistance determined by two alleles at one locus. Pretreatment allelic frequencies are maintained within the model by heterozygote disadvantage which results in suprapopulation mortalities. The model predicts that alternating anthelmintics with different modes of action may be a less effective resistance management strategy than administering the same drugs simultaneously. The simple simulation model described by Martin and McKenzie (1990b) assumes a constant biotic potential and determines the contribution made to the next generation by recruitment from the suprapopulation and post-treatment infrapopulation. Table III summarizes some of the findings produced by the model which highlights the importance of a high suprapopulation/post treatment infrapopulation ratio in delaying the rate of development of resistance. The findings from the model support the view that the onset of resistance in temperate zones, where a very high proportion of the total population is normally in refugia, may be delayed by ensuring that dosing removes all heterozygous resistant individuals.

DELAYING T H E ONSET OF RESISTANCE Observations from field and laboratory studies on the development of resistance and the results from models of anthelmintic resistance all point towards the need for preventive strategies aimed at delaying the onset of resistance. Preventive strategies inevitably incorporate minimal chemoprophylaxis, thus minimizing the number of parasite generations exposed to a drug, and also seek to maximize efficacy, thus effectively rendering the genes for resistance recessive. Preventive strategies naturally require a sound knowledge of the epidemiology of the target para-

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site species and are heavily reliant u p o n an integrated a p p r o a c h to control, using means o t h e r than c h e m o t h e r a p y such as grazing m a n a g e m e n t , to limit h o s t / p a r a s i t e contact and thus r e d u c e the n e e d for f r e q u e n t treatment. This a p p r o a c h has b e e n used successfully in d e v e l o p i n g epidemiologically based regimes (Dash et al., 1985) to conserve the efficacy o f ivermectin ill areas of Australia where Haemonchus is the p r e d o m i n a n t parasite. Unfortunately, these control regimes, which i n c o r p o r a t e salicylanilides such as closantel, are tailored tO suit h a e m a t o p h a g o u s parasites and thus are not universally applicable. W h e r e anthelmintics are used in preventive strategies clearly they must have a high efficacy and remove heterozygous resistant genotypes. C o m b i n a t i o n s of anthelmintics offer o n e means of maximizing efficacy and have been used in studies on the d e v e l o p m e n t o f resistance. In o n e e x p e r i m e n t a l study which used either a benzimidazole, levamisole or a c o m b i n a t i o n of both drugs, no resistance was evident after six generations of parasite were exposed to the c o m b i n a t i o n t r e a t m e n t whereas resistance a p p e a r e d to the single drugs within three to four generations of application (Martin et al., 1990). T h e authors o f a n o t h e r study in Australia also suggested that two drug c o m b i n a t i o n s using b e n z i m i d a z o l e / l e v a m i s o l e / o r g a n o p h o s p h o r u s c o m b i n a t i o n s could be used in preventive strategies (Anderson et al., 1990). Because effective preventive strategies must allow for tile flfll genotypic range that may be f o u n d within the parasite population, when genotypic restriction, often a characteristic feature o f individual studies, occurs it suggests that caution be used when a t t e m p t i n g to extrapolate fi'om them. Genotypic restriction xnay a c c o u n t for differences in rate o f selection o f resistance that o c c u r on farms using the same t r e a t m e n t regimes a n d / o r the rate at which resistance to individual d r u g families emerges. Given that little or no influx occurs t h r o u g h host m o v e m e n t then tile limited capacity that n e m a t o d e s u p r a p o p u l a t i o n s have to migrate ensures relatively stable genetic b a c k g r o u n d s for the parasites on an individual farm. This relatively narrow genetic base for selection was acknowledged as an i m p o r t a n t factor in a 5-year study c o n d u c t e d on pastures c o n t a m i n a t e d with Haemonchus and 7: colubdformis in Australia (Waller et aL, 1989). T h e study used all three b r o a d spectrum anthehnintics in a variety o f different t r e a t m e n t strategies and f o u n d that suppressive regimes selected only benzimidazole resistance. Within the confines o f the study, albendazole had a h i g h e r efficacy and delayed the d e v e l o p m e n t of resistance and thiabendazole was shown to counterselect against levamisole resistance. Two o t h e r interesting observations to e m e r g e fl-om the study were, firstly, that anthehnintic rotation at yearly intervals was m o r e beneficial than rotating after each treatment. Secondly, there was n o evidence that treating animals and moving them to minimally c o n t a m i n a t e d pasture p r o d u c e d m o r e resistance than set stocking, when the same n u m b e r of treatments were given in each regime. T h e two species featured in that study, Haemondms and 7: cotum&4formi.~, are relatively f e c u n d species, an attribute which clearly can affect the rate o f developm e n t of resistance. During the initial selection process, even when pastures car W only a small population o f susceptible genotypes, the n u m b e r s of adults recruited from the p o p u l a t i o n on pasture is always likely to e x c e e d those surviving treatment, and thus susceptible genotypes with a high biotic potential are at an advantage. U n d e r the same circumstances, the susceptible genotypes o f less f e c u n d

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species, particularly those like Teladorsagia that have stereotyped egg outputs (Michel, 1969;Jackson & Christie, 1979), gain less adva.ntage. Despite the a p p a r e n t drawbacks i n h e r e n t in the use o f minimally c o n t a m i n a t e d pasture, grazing m a n a g e m e n t is still a valuable strategy which can be a d o p t e d in o r d e r to r e d u c e the f r e q u e n c y of treatment. T h e benefits resulting f r o m suprap o p u l a t i o n reduction, gained at the expense of p r o d u c i n g minimally contamin a t e d pasture, have to be weighed against the potential risk of increasing selection for resistance. In particular, the practice o f treating animals grazing minimally c o n t a m i n a t e d pastures, in an effort to maintain a r e d u c e d level o f c o n t a m i n a t i o n , offers little or no sustainable benefit and will inevitably accelerate the rate o f selection of resistance. Parasite infi'apopulation dynamics must also be taken into consideration in the d e v e l o p m e n t of preventive strategies for different host species. In adult fibre-producing goats, hypobiotic larvae a p p e a r to play an i m p o r t a n t role in the process o f selection o f resistance (Jackson, u n p u b l i s h e d data). Multiple treatments given d u r i n g the course o f a grazing season will expose these larvae to an intense selection pressure and may result in a highly refractory p o p u l a t i o n at the e n d o f the grazing season. U n d e r these circumstances it is i m p o r t a n t to p r e v e n t the maturation of these larvae and thus limit their potential to transmit the genes for resistance. Since m a t u r a t i o n of inhibited larvae is most likely to o c c u r d u r i n g the periparturient relaxation o f host immunity, it is i m p o r t a n t that treatments administered at this time are highly effective. Although their efficacy u n d e r UK conditions remains to be tested, c o m b i n a t i o n (Martin et al., 1990; A n d e r s o n et al., 1990) or split dose strategies (Sangster et al., 1991), which are known to maximize efficacy, may well be o f value to goat owners at such times. Goat owners o p e r a t i n g a slow a n t h e h n i n t i c rotation might usefully consider designating o n e o f these early season treatments as the transitional t r e a t m e n t and d r e n c h their does simultaneously with both drugs.

CONTROL OF RESISTANCE

Environmentally and or immunologically based control strategies which seek to limit h o s t / p a r a s i t e contact have an obvious application in the avoidance and mana g e m e n t of a n t h e h n i n t i c resistance alongside properly directed c h e m o t h e r a p y . T h e m a n a g e m e n t o f s i n g l e and multiple resistance imposes obvious restraints u p o n c h e m o p r o p h y l a x i s since it dictates which families may be used with certainty o f efficacy. Moreover, if selection o f resistance to these families is to be avoided then it is clearly p r u d e n t also to limit the fi'equency o f application. T h e tact that very few p r o d u c e r s routinely screen for anthelmintic resistance, c o u p l e d with the relatively p o o r sensitivity of the most c o m m o n l y used in vivo screening methods, ensures that most cases o f resistance are not d e t e c t e d at an early stage. This reduces the likelihood o f reversion o c c u r r i n g and thus, to avoid increasing levels o f resistance a n d / o r the n u m b e r s o f resistant species, conventional wisdom suggests p r o l o n g e d or total withdrawal of the selecting family or families. Whilst there can be no d o u b t about the wisdom o f this advice in the mana g e m e n t of single family resistance, the e m e r g e n c e o f multiple resistance in

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Australia has focused attention upon the use of anthelmintics against which resistance has already been selected, in the management of resistance. The efficacy of these 'selected' drugs applied either at the recommended or at a higher dose rate has been tested either singly, often in extended treatment application (Anderson, 1990; Sangster et aL, 1991), or used in combination with another family or families (Anderson et aL, 1990). The therapeutic value of a combination of albendazole and levamisole was proven in a study in Australia in a trial covering 22 properties over 70% of which had either benzimidazole or levamisole resistance. A single drench at the recommended dose rate reduced egg counts by at least 95% on half of the farms and a double dose rate proved to be effective on over 80% of the farms (Anderson et aL, 1990). Another comparative study in Australia examined the development of resistance and performance of animals given either a bolus containing a benzimidazole or oral treatments with ivermectin or a benzimidazole. The study started with 20% heterozygous benzimidazole-resistant strains of Teladorsag~a and T. colubriformis, and faecal egg count reduction tests showed that in the bolus treated group efficacy over 3 years was reduced from 94-100% to 50-65%. Despite this marked reduction in efficacy, productivity of the bolustreated animals was not less than that of orally treated groups, including those animals treated with ivermectin, but was significantly higher than untreated controls (Anderson, 1990). The risks associated with the re-introduction of 'selected' drugs for therapeutic and prophylactic purposes are influenced largely by the pathogenicity and fecundity of the prevailing resistant species and the extent of any increase in resistance that results from further exposure. Where the predominant resistant species have high biotic potential and also are highly pathogenic, as is the case with Haemonchus, then clearly the risks associated with reintroduction are high. Given that the production and welfare of treated animals are not compromised then it may even be possible, under carefully monitored circumstances, to reintroduce 'selected' drugs into slow chemoprophylactic rotations, particularly when resistance involves less pathogenic species with a low biotic potential such as Teladorsagia. The risks associated with the reintroduction of drugs require assessment against the main benefit, the alleviation of pressure for developing multiple resistance. The possibility of reintroducing 'selected' drugs for therapeutic and prophylactic purposes clearly requires further investigation in other ecological zones and management systems with different resistant species and ecotypes. An entirely different approach to the management of resistance has been attempted in South Africa where an attempt has been made to reintroduce susceptible genes into the suprapopulation (Van Wyk, 1990). The study showed that it was possible to overwhelm a benzimidazole-resistant strain of H. contortus on pasture using donor sheep infected with a susceptible strain of the same species. The degree of genetic replacement was assessed using tracer lambs and the best success was achieved in spring, at a time when the pasture was contaminated with worm eggs but was not infective, thus enabling the susceptible population to accumulate when the resistant population could not replicate. This novel strategy may have a limited application, suiting certain climates and those species with high biotic potential but unless the genes for resistance are totally eradicated, the potential for rapid reselection must remain high. This approach is unlikely to be

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applicable in temperate climates due to differences in suprapopulation dynamics and the low fecundity of some of the important resistant species such as Teladorsagia.

CONCLUSIONS

Anthelmintic resistance provides a clear example of the adaptability of metazoan parasites and illustrates graphically the risks inherent in intensively applied control strategies which exert a high selection pressure. Examples of parasite adaptability also exist within other approaches to the control of nematodoses. The use of alternate grazing systems incorporating young calves has led to the selection of Nematodirus battus populations adapted to both young cattle and sheep (Coop et al., 1988) and has resulted in disease caused by this species in calves (Armour et al., 1988). Parasite adaptability appears to be a highly pertinent factor influencing established approaches to the control of parasitoses and certainly deserves consideration during the development of any new approaches to control. Antibodymediated immunological approaches to parasite control, particularly if based upon a limited antigen/target protein repertoire appear, because of the extended duration of antibody responses, to have the potential to select very strongly for parasite genotypes with a minimal reliance upon the target proteins. Parasite adaptability has undoubtedly influenced the evolution of immunoresponsiveness and helps to account for the complexity of the effector mechanisms seen in studies on naturally acquired immunity (Miller, 1984). It appears that, in future, sustainable control strategies for helminthoses may require an integrated approach incorporating environmental management, immuno- and chemoprophylaxis in order to minimize the pressure for parasite adaptation. Unfortunately, such approaches are complex and climate- and parasite-specific and are thus less easily developed than strategies based on a single means of control. However, one of the main lessons to emerge from studies on anthelmintic resistance must be that whilst the need for intensive production systems remains, the costs of developing integrated approaches for control of parasitoses must be met in order to achieve acceptable standards of animal health and welfare.

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