Anthelmintic resistance: The state of play revisited

Life Sciences 79 (2006) 2413–2431www.elsevier.com/locate/lifescie

Mini review

Anthelmintic resistance: The state of play revisited

Abdul Jabbar a,⁎, Zafar Iqbal a, Dominique Kerboeuf b, Ghulam Muhammad c,Muhammad N. Khan a, Musarrat Afaq a

a Chemotherapy Laboratory, Department of Veterinary Parasitology, University of Agriculture, Faisalabad-38040, Pakistanb MultiResistances et Antiparasitic drugs, IASP-213, INRA-Tours, 37380 Nouzilly, France

c Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad-38040, Pakistan

Received 14 October 2005; accepted 14 August 2006

Abstract

Helminthosis is one of the major constraints in the successful wool and mutton industry throughout the world. Anthelmintic Resistance (AR) issaid to have been established when previously effective drug ceases to kill exposed parasitic population at the therapeutically recommendeddosages. Anthelmintic resistance is almost cosmopolitan in distribution and it has been reported in almost all species of domestic animals and evenin some parasites of human beings. Some of the most important species of parasites of small ruminants in which AR has been reported include:Haemonchus spp., Trichostrongylus spp. Teladorsagia spp., Cooperia spp. Nematodirus spp., and Oesophagostomum spp. All the major groupsof anthelmintics have been reported for development of variable degrees of resistance in different species of gastrointestinal nematodes. This paperdescribes the global scenario of prevalence and methods used for detection of AR in small ruminants. Different mechanisms and contributoryfactors for the development of AR are discussed. Various options and alternate strategies for the control and/or delay in the onset of AR aresuggested in the light of available information.© 2006 Elsevier Inc. All rights reserved.

Keywords: Resistance; Benzimidazole; Levamisole; Ivermectin; Gastrointestinal nematodes; Sheep; Goat

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2414Prevalence of anthelmintic resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2414

Geographic distribution of parasites showing anthelmintic resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2416Drugs to which resistance has been developed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2416

Factors contributing towards development of resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2416Treatment frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2416Use of anthelmintics in sub-optimal doses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2417Targeting and timing of mass treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2417Single-drug regimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2417Other factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2417

Mechanisms of anthelmintic resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2418Benzimidazoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2418Levamisole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2418Ivermectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2418

Genetics of nematodes and anthelmintic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2419Detection of resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2419

⁎ Corresponding author. Tel.: +92 41 9201106.E-mail address: [email protected] (A. Jabbar).

0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.lfs.2006.08.010

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In vivo tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2420Faecal egg count reduction test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2420Critical anthelmintic test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2420The controlled anthelmintic efficacy test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2420

In vitro tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2421Egg hatch test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2421Larval development assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2421Adult development test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2421Larval paralysis test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2422Larval motility test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2422Adult migration inhibition test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2422Colorimetric assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2422Polymerase chain reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2422

Control of resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2423Delaying the onset of anthelmintic resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2423

Anthelmintic unexposed population of parasites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2423Modelling of anthelmintic use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2423Adoption of strict quarantine measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2423

Alternate strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2424Resistant animal breeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2424Grazing management and anthelmintic treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2424Nutrition and parasite interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2424Antiparasitic vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2424Botanical dewormers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2424Biological control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2425

Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2425Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2425References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2425

Introduction

Despite remarkable achievements in the discovery anddevelopment of anthelmintic drugs, nematode parasitic diseaseremains one of the greatest limiting factors to successful, andsustainable ruminant livestock production, worldwide (Perryand Randolph, 1999). Nematodosis can cause direct losses dueto drop in production and deaths of animals. Furthermore, mostof the economic losses are due to sub-clinical effects which arenot immediately noticed by the owner. Lanusse and Prichard(1993) estimated that about 1.7 billion US$ is spent annuallyworldwide to combat helminth parasites in cattle. Although, theamount spent on small ruminants is much less, it is still quitesubstantial. In Australia, the estimated cost to control worms insheep is between 220 (McLeod, 1995) to 500 millions US$(Emery and Wagland, 1991). Considering the reported largeproblems in the sheep industry in South and Central Americaplus South Africa due to AR, the costs of treatment in thesecountries would be very high.

The anthelmintic resistant gastro-intestinal (GI) nematodepopulations constitute a major problem especially in smallruminants not only in the subtropics and tropics, but also in aserious threat to livestock in rest of the world (Conder andCampbell, 1995; Waller, 1997; Sangster, 1999). Anthelminticresistance in nematode parasites of almost all species of animalsis now a firmly established phenomenon, particularly in warmtemperate or tropical regions of the world (Waller and Prichard,1985). Resistance is considered to have been established whenpreviously effective drug ceases to kill exposed parasitic

population at the therapeutically recommended dosages (Pri-chard et al., 1980; Jackson, 1993). The existence of AR came tolight in the mid-1950s as a result of the failure of phenothiazineto control haemonchosis in a flock of sheep kept at a researchfarm in Kentucky, USA (Drudge et al., 1957). The developmentof resistance by nematodes to broad-spectrum anthelmintics isof particular concern. Currently, three different chemicalgroups, i.e., benzimidazole (BZs), imidazothiazoles andavermectins are commonly used for deworming. A varyingdegree of resistance in nematode populations against theseanthelmintics has been widely reported throughout the world(Prichard et al., 1980; Jackson et al., 1987; Prichard, 1990;Jackson, 1993; Besier and Love, 2003; Coles, 2005). Thedevelopment of AR, therefore, has resulted in lowered animalproductivity due to heavy nematode burden.

This paper reviews prevalence of AR in gastrointestinalnematodes (GINs) of small ruminants against commonly usedanthelmintics. It also describes the factors contributing towardsAR, mechanisms of development of AR, methods for thedetection of resistance and some possible solutions to controlthe development of AR.

Prevalence of anthelmintic resistance

Many of the earliest reports of ruminant nematode strainsresistant to broad-spectrum anthelmintics emanated from thesouthern hemisphere and usually involved species with a highbiotic potential such as Haemonchus (H.) contortus and Tri-chostrongylus (T.) colubriformis. The rate of emergence of

Table 1Anthelmintic resistance reported in different parts of the world (selected references)

Species/genus of nematodes Resistant to anthelmintic Country Reference(s)

O. circumcincta TBZ, LEV, Morantel Australia Hall et al. (1979)H. contortus TBZ Australia Webb et al. (1979)H. contortus BZ, Non-BZ,

Organophosphorus AnthelminticsAustralia Green et al. (1981)

Haemonchus, Trichostrongylus,Ostertagia spp.

FEN, LEV Australia Barton et al. (1985)

T. colubriformis OXF, LEV Australia Dash (1986)Haemonchus, Trichostrongylus,

Ostertagia, NematodirusTBZ, LEV Australia Edwards et al. (1986)

Trichostrongylus, Ostertagia spp. TBZ, LEV, ALB, OXF, Morantel Australia Ottaway and Webb (1986)H. contortus, Trichostrongylus,

Ostertagia spp.TBZ, LEV Australia Webb and Ottaway (1986)

Sheep nematodes TBZ, LEV Australia Anderson et al. (1988)H. contortus, T. colubriformis,

O. circumcinctaOXF, LEV, Closantel, Morantel Australia Love et al. (1992)

H. contortus BZ Belgium Dorny et al. (1993)T. circumcincta TBZ, IVM, LEV Brazil Amarante et al. (1997)O. circumcincta LEV Denmark Bjorn et al. (1992)Ostertagia, Trichostrongylus spp. T5BZ, LEV, IVM Denmark Maingi et al. (1997)Goat nematodes BZ France Kerboeuf and Hubert (1985)H. contortus BZ France Gruner et al., 1986;

Kerboeuf et al., 1989;Hubert et al., 1991

H. contortus, Trichostrongylus,Ostertagia spp.

BZ, LEV France Kerboeuf et al. (1988)

H. contortus BZ Germany Duwel et al. (1987)Gastrointestinal nematodes TBZ, MBZ, FEN, ALB,

OXF, IVM, LEV, Febantel,Pyrantel Tartrate

Germany Bauer et al. (1988)

Nematodes BZ, LEV, Morantel Greece Papadopoulos et al. (1994)Nematodes BZ Ireland O'Brien et al. (1994)H. contortus BZ India Yadav (1990)H. contortus BZ, LEV, IVM, Thiophanate, Morantel, Closantel India Uppal et al. (1992)H. contortus FEN, LEV, IVM,

Closantel, MorantelIndia Yadav et al. (1993)

Gastrointestinal nematodes ALB, LEV, IVM India Gill (1996)H. contortus FEN, LEV, IVM, Morantel India Singh and Yadav (1997)Sheep and goat nematodes BZ Italy Genchi (1994)H. contortus Trichostrongylus spp. BZ, FEN, LEV Kenya Maingi (1991)Gastrointestinal nematodes BZ, LEV, IVM Kenya Shivairo et al. (1996)H. contortus Thiophanate, Closantel, ALB, LEV, IVM Kenya Waruiru et al. (1997)H. contortus, Trichostrongylus,

Oesophagostomum spp.BZ, LEV, IVM, RAF Kenya Waruiru et al. (1998)

Goat nematodes TBZ Malaysia Rahman (1993)H. contortus, T. colubriformis BZ, LEV, IVM,

Closantel, MoxidectinMalaysia Sivaraj et al. (1994)

Trichostrongylus spp BZ Netherlands Boersema et al. (1987)H. contortus, C. curticei, Ostertagia,

Trichostrongylus spp.OXF, IVM, LEV Netherlands Borgsteede et al. (1997)

H. contortus, O. circumcincta BZ, LEV, IVM Netherlands Várady and Čorba (1999)Sheep nematodes BZ, Non BZ New Zealand Kettle et al. (1982)Cooperia oncophora OXF New Zealand Jackson et al. (1987)H. contortus, Trichostrongylus spp. BZ, LEV, IVM New Zealand McKenna et al. (1990)Sheep nematodes BZ, LEV, IVM New Zealand Leathwick et al. (1996)Trichostrongyles BZ North West Cameroon Ndamukong and Sewell (1992)Sheep gastrointestinal nematodes BZ, LEV Pakistan Afaq (2003)H. contortus OXF Pakistan Jabbar (2003)Sheep gastrointestinal nematodes OXF, LEV Pakistan Saddiqi et al. (in press)Trichostrongylus spp. BZ Slovakia Praslicka (1994)Ostertagia, Trichostrongylus spp. BZ, LEV, TMZ Slovakia Corba et al. (1998)H. contortus IVM, BZ, CLOST, Rafoxanide South Africa van Wyk et al. (1997)H. contortus, Ostertagia,

Trichostrongylus spp.BZ, LEV IVM, Closantel Southern Latin America, Brazil Echevarria et al. (1996)

(continued on next page)

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Table 1 (continued)

Species/genus of nematodes Resistant to anthelmintic Country Reference(s)

H. contortus Trichostrongylus,Oesophagostomum

BZ, LEV, IVM South Latin America, Argentina Eddi et al. (1996)

H. contortus Trichostrongylus,Oesophagostomum

BZ, LEV Southern Latin America Maciel et al. (1996)

H. contortus, Ostertagia spp. BZ, LEV, IVM Southern Latin America, Uruguay Nari et al. (1996)T. circumcincta Netobimin, BZ Spain Requejo-Fernandez et al. (1997)H. contortus TBZ United Kingdom Cowthorne and Whitehead (1983)O. circumcincta TBZ United Kingdom Britt and Oakley (1986)H. contortus FEN, IVM United Kingdom Scott et al. (1989)H. contortus, O. circumcincta United Kingdom Taylor and Hunt (1989)H. contortus, O. circumcincta BZ, IVM United Kingdom Jackson et al. (1991)O. circumcincta BZ United Kingdom Mitchell et al. (1991)Sheep nematodes BZ United Kingdom Hong et al. (1992)Sheep and goat nematodes BZ, LEV, IVM United Kingdom Hong et al. (1996)Sheep nematodes BZ, LEV, IVM United Kingdom Coles (1997)H. contortus TBZ United States of America Theodorides et al. (1970)H. contortus, O. circumcincta TBZ United States of America Miller and Baker (1980)H. contortus FEN, LEV, IVM, Pyrantel pamoate United States of America Uhlinger et al. (1992)H. contortus BZ, LEV, FEN, RAF Zimbabwe Boersema and Pandey (1997)

C. = Cooperia; H. = Haemonchus; O. = Ostertagia; T. = Trichostrongylus; T. = Teladorsagia; BZ = Benzimidazole; MBZ =Mebendazole; FEN = Fenbendazole; TBZ =Thiabendazole; ALB = Albendazole; OXF = Oxfendazole; LEV = Levamisole; IVM = Ivermectin; RAF = Rafoxanide; TMZ = Tetramizole; CLOST = Closantel.

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resistance appears to vary geographically in accordance with theprevailing climate, parasite species and treatment regimesadopted in the region. Although the rate of emergence ofresistant strains has generally been slower in temperate zones inthe northern hemisphere, the prevalence of resistance is alsoincreasing throughout Europe (Borgsteede and Stallinga, 1990;Bjorn et al., 1990; Taylor et al., 1990; Scott et al., 1989; Walleret al., 1990) and the rest of the world (Anderson et al., 1988;Singh and Yadav, 1997).

Geographic distribution of parasites showing anthelminticresistance

Anthelmintic resistance in the field is usually noticed whenworm control policies fail dramatically. Throughout the world,resistance has been detected most commonly among the GINsof sheep and goats, notably H. contortus and Teladorsagia (T.)circumcincta, although parasites belonging to the Trichostron-gylus, Cooperia, and Nematodirus genera have also reportedlydeveloped AR (Taylor and Hunt, 1989). Prevalence of AR indifferent GINs dwelling in small ruminants along with reportedcountry has been presented in Table 1.

Drugs to which resistance has been developed

Resistance has been recorded in many countries throughoutthe world against drugs in all of the three broad-spectrumfamilies, the BZs, avermectins and imidazothiazoles, which arecommonly used by the livestock industry to control nemato-dosis (Prichard et al., 1980; Coles, 1986; Waller, 1987;Prichard, 1990). Resistance has been recorded also in drugswith a narrower spectrum of activity such as the salicylanilides(Jeannin et al., 1990; Scott and Armour, 1991). The majoranthelmintic drugs against which AR has been reported

include: phenothiazine (Drudge et al., 1957), thiabendazole(TBZ) and other BZs (Drudge et al., 1964; Green et al., 1981;Waruiru et al., 1998; Zajac and Gipson, 2000), ivermectin(IVM) (van Wyk and Malan, 1988; Echevarria and Trindale,1989; van Wyk et al., 1989) and levamisole (LEV) (Gillhamand Obendorf, 1985). It is alarming that GINs of smallruminants have developed resistance against all major groupsof anthelmintics which compel to think about alternatives to useof chemicals against GINs.

Factors contributing towards development of resistance

Modern anthelmintics are used at an efficiency of around99% against susceptible strains. A small number of survivingworms, which are the most resistant component of thepopulation, then contaminate the pasture with a majority ofresistant offsprings for subsequent generations which lead todevelopment of AR due to selection pressure. The rate ofdevelopment of resistance is influenced by many factors whichcan be classified as genetic, biological or operational. The mostimportant are the operational factors because they can bemanipulated by the farmer and form the bases of resistancemanagement programmes. However, it is necessary to under-stand the genetic and biological factors in order to arrive at thecorrect operational procedures. Of the several factors contrib-uting to AR, significant ones are described here.

Treatment frequency

It has been observed that frequent usage of the same group ofanthelmintic may result in the development of AR (Martin et al.,1982; Barton, 1983; Coles, 1986; Waller, 1987; Taylor andHunt, 1989). There is evidence that resistance develops morerapidly in regions where animals are dewormed regularly.

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Anthelmintic resistance in H. contortus has been reported insome humid tropical areas where 10 to 15 treatments per yearwere used to control this parasite in small ruminants (Dorny etal., 1994). Drug resistance, however, can also be selected atlower treatment frequencies, especially when the same drug isused over many years. Coles et al. (1995) have reported thedevelopment of AR even when only two or three treatmentswere given annually.

Use of anthelmintics in sub-optimal doses

Underdosing is generally considered an important factor inthe development of AR (Edwards et al., 1986) because subtherapeutic doses might allow the survival of heterozygousresistant worms (Smith, 1990). Several laboratory experimentshave shown that underdosing contributes to the selection ofresistant or tolerant strains (Egerton et al., 1988; Hoekstra etal., 1997). Moreover, variation in bioavailability in differenthost species also is crucial for making a decision about correctdose. Some indirect field evidence further supports thisconclusion. For an example, the bioavailability of BZ andLEV is much lower in goats than in sheep, resultantly thosegoats should be treated with dosages 1.5 to 2 times higher (thesingle dose is much less inferior than “sub-optimal”, it is rathernear half the dose necessary for goats) than those given tosheep (Hennessy, 1994). For many years, however, sheep andgoats were given the same anthelmintic doses. The fact thatAR is very frequent and widespread in goats may be a directconsequence of difference in metabolism of drugs. Consideringthis extrapolation, modeling exercises suggest that the fieldsituation of AR is not always simple (Smith et al., 1999).Depending on the initial frequency of the resistance alleles,there might be a range of dose levels where underdosingpromotes resistance and a range of dose levels where itactually impedes resistance. Although, further research on theimpact of underdosing on the development of resistance isnecessary, current knowledge advises against the use ofsubcurative dosages.

To reduce the costs of anthelmintic treatment in developingcountries, the use of lower dosages than the recommendedtherapeutic ones has been advocated (Warren et al., 1993). Suchpractices should clearly be avoided. Most of the currentlyapplied anthelmintics are in fact subcurative in at least part ofthe population. Additionally, there are a number of species ofnematodes which are present as mixed infection in animalsthroughout the world which respond to different groups ofanthelmintics differently due to the irregular susceptibility ofthese species to a given anthelmintic. This is consideredacceptable for morbidity control, but in the long run suchstrategies may contribute to the development of AR as well.Furthermore, generic products of substandard quality, repackedand/or reformulated products, and expired drugs are wide-spread in pharmacies and general markets. The presence ofpoor-quality drugs has been documented in human as well as inveterinary medicine (Monteiro et al., 1998; Shakoor et al.,1997). Over-the-encounter availability of drugs includinganthelmintics in developing countries like Pakistan may also

be an important factor in the development of AR (personalcommunication).

Targeting and timing of mass treatment

Prophylactic mass treatments of domestic animals havecontributed to the widespread development of AR in helminths.Computer models indicate that the development of resistance isdelayed when 20% of the flock is left untreated (Barnes et al.,1995; van Wyk, 2001) but it needs confirmation through ex-perimentation. This approach would ensure that the progeny ofthe worms surviving treatment will not consist only of resistantworms. Given the well-known over-dispersed distribution ofhelminths, leaving a part of the group untreated, especially themembers carrying the lowest worm burdens, should notnecessarily reduce the overall impact of the treatment.

In worm control in livestock, regular moving of the flocks toclean pastures after mass treatment and/or planning toadminister treatment in the dry seasons is a common practiceto reduce rapid reinfection. However, these actions result in thenext helminth generation that consists almost completely ofworms that survived therapy and, therefore, might contribute tothe development of AR (Smith, 1990; Taylor and Hunt, 1989).For an example, Coles et al. (1995) reported problem of AR inthe helminths of sheep and goats on some small Greek islandswhich suffered from extended drought with, in contrast, no ARdeveloped under similar management and deworming practiceson the mainland.

Single-drug regimens

Frequent and continuous use of a single drug leads to thedevelopment of resistance. For example, a single drug, whichis usually very effective in the first years, is continuously useduntil it no longer works (Pal and Qayyum, 1996). In a surveyof sheep farmers in the United States, Reinemeyer et al.(1992) found that one out of every two flocks was dosed witha single anthelmintic until it failed. Long-term use of LEV incattle also led to the development of resistance, although theannual treatment frequency was low and cattle helminthsseemed to develop resistance less easily than do worms insmall ruminants (Geerts et al., 1987). Frequent use of IVMwithout alternation with other drugs has also been reported asthe reason for the fast development of resistance in H.contortus in South Africa and New Zealand (van Wyk et al.,1989; Shoop, 1993).

Other factors

There are some other factors which can also contributetowards the development of AR including introduction ofresistant parasites by means of animals transported from countryto country (Várady et al., 1994) and keeping the sheep and goatstogether (Jackson, 1993). Furthermore, factors like nematodesin refugia, gene frequency in unselected nematodes, genetics ofAR and biological fitness of unselected worms may alsocontribute in the development of AR (Coles, 2005).

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Mechanisms of anthelmintic resistance

Benzimidazoles

The best known mechanism of resistance is the one to BZs.The BZs exert their anthelmintic activity by binding to β-tubulin,which interfereswith the polymerization of themicrotubuli. Someauthors (Beech et al., 1994; Roos et al., 1995) have shown thatthere is an extensive polymorphism of the β-tubulin gene insusceptible H. contortus populations. Roos et al. (1995) provedthat selection for resistance to BZs is accompanied by a loss ofalleles at the locus of β-tubulin isotype-1. It has been discoveredthat resistance to BZs is correlated with a conserved mutation atamino acid 200 in β-tubulin isotype 1 (with Phenylalanine beingreplaced by Tyrosine) (Kwa et al., 1994; Robinson et al., 2002).The samemutationwas shown to occur in BZ-resistant fungi suchas Aspergillus nidulans and Venturia inaequalis (Jung et al.,1992). The functional importance of this amino acid substitutionwas shown by heterologous expression of the β-tubulin isotype 1(isolated from BZ-susceptible H. contortus) in BZ-resistantCaenorhabditis elegans. Expression of the H. contortus genealtered the phenotype of transgenic C. elegans from resistant tosusceptible. Conversely, when Phe was replaced by Tyr at aminoacid position 200 of this gene by in vitro mutagenesis, thereverting activity was lost (Kwa et al., 1995).

Another resistance mechanism was identified in some H.contortus populations showing higher levels of resistance, inwhich deletion of the β-tubulin isotype 2 locus was shown(Roos et al., 1995). However, Beech et al. (1994) could notconfirm this in other BZ-resistant H. contortus populations.These authors also showed that changes in allele frequenciesrather than novel rearrangements induced by exposure to thedrug explained changes associated with BZ resistance. A similarstepwise selection of BZ resistance also occurs in some T.colubriformis and T. circumcincta populations (Elard et al.,1996; Grant and Mascord, 1996). Since specific BZ resistanceseems to be due to similar point mutations in several fungi andnematodes of veterinary importance, it is not unlikely that itwould be relevant for resistance in human nematodes as well.

Furthermore, Kerboeuf et al. (1999) provided indirect evidencethat P-glycoproteins (P-gp) also play a role in BZ resistance in H.contortus. P-gp are involved inmultidrug resistance inmammaliantumor cells, Leishmania, and Plasmodium and in resistance totoxic compounds in C. elegans. Rhodamine 123, a P-gp transportprobe, associated with the reversal agent verapamil (an inhibitor ofmultidrug resistance-associated proteins), gave significantly higherlevels of fluorescence in eggs fromH. contortus resistant toBZ andIVM than in susceptible eggs. These results confirm those obtainedwith biological drug assays using both anthelmintics andverapamil. Thus, Beugnet et al. (1997) showed that addition ofverapamil to eggs partially reversed resistance to thiabendazoleand this was attributed to the presence of P-glycoproteins of whichrole in resistance was further demonstrated (Kerboeuf et al., 2003).However, Kwa et al. (1998), using a P-gp gene probe from H.contortus, were not able to correlate polymorphism to any of the(multi) drug resistances examined in different H. contortuspopulations. It should be noticed that the DNA used by Kwa et

al. (1998) was prepared from pooled L3 larvae and not fromindividual parasites, so that no estimates of allele frequencies couldbemade (Anderson et al., 1998). Since at least 14 P-gp genes seemto be present in C. elegans, it is also possible that P-gp other thanthose characterized by Kwa et al. (1998) or multidrug resistance-associated proteins might be involved in drug resistance.

Recently, Riou et al. (2005) have studied the changes inmembrane environment (eggshells) of H. contortus eggs duringthe embryonation by fluidity measurements and their effects onnon-specific mechanisms of resistance to anthelmintics. Theresults obtained showed that the embryonation induced asignificant and gradual increase in eggshell fluidity which maybe associated with an increased resistance to anthelmintics.Riou et al. (2003) also showed for the first time the role ofcholesterol in modulating drug resistance in nematodes and theassociation between P-gp activity and cholesterol concentrationin eggs.

Levamisole

Levamsiole is cholinergic agonist with a selective action onnematode receptors. The mechanism of resistance to LEV hasnot yet been fully elucidated. Sangster (1996) reviewed thepharmacology of LEV resistance. It is thought to be causedeither by a reduction of the number of nicotinic acetylcholin-esterase receptors or by a decreased affinity of these receptorsfor the drug. Hoekstra et al. (1997) were able to clone the geneHca 1, encoding the nicotinic acetylcholinesterase receptorfrom H. contortus. Although, polymorphism at the amino acidlevel could be demonstrated, these authors could not findevidence that alleles at this locus were involved in selection forresistance to LEV. A similar gene, tar-1 was identified on the Xchromosome in T. colubriformis (Wiley et al., 1997). Although,statistical comparison of allele frequencies from individual maleand female worms was consistent with sex linkage of tar-1, nocorrelation was found with LEV resistance status. The exactmechanism of LEV resistance still needs to be researched toreach ultimate conclusion.

Ivermectin

IVM and other macrocyclic lactones affect GINs by causingstarvation and/or paralysis by opening chloride channels, whichare thought to be associated with α-subunits of glutamate-gatedion channels located on muscles of the pharynx and possibly thesomatic musculature (Sangster, 1996). Rohrer et al. (1994)compared IVM-resistant and -susceptible H. contortus popula-tions and found that resistance is not due to an alteration in thebinding of IVM to glutamate gated chloride channel receptors.Nevertheless, Blackhall et al. (1998) did report that one allele ofthe putative α-subunit gene is associated with resistance to thedrug. These workers reported considerable genetic variation of aP-gp locus in H. contortus. In several drug-selected strains ofthe parasite, selection for the same allele was observed. Usingdifferent approaches, Xu et al. (1998) and Sangster et al. (1999)came to the conclusion that P-gp might be involved in resistanceto IVM in this helminth species.

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Other mechanisms of resistance may be present as well, assuggested by Gill et al. (1998) and Gill and Lacey (1998). Thelatter workers described five possible types of resistance to IVMin H. contortus based on different behavior in in vitro tests(larval development assay and L3 motility tests), differentsensitivity to paraherquamide (an anthelmintic with a com-pletely different structure and different binding sites from IVM),and different inheritance (in at least two of the five resistancetypes). Gill and Lacey (1998) also suggested that themechanism of resistance to IVM might be different from onespecies of helminth to another, because the critical eventsleading to expulsion have been shown to be different, e.g., whenOstertagi ostertagi is compared to H. contortus and T.colubriformis.

Genetics of nematodes and anthelmintic

Nematode parasite populations are genetically heteroge-neous and thus able to respond to selective pressures, i.e.,anthelmintic drugs (Grant, 1994). Widespread drug pressurewill favour and select parasite lines carrying tolerance orresistance alleles. The rate at which resistance spreads in theparasite population depends on many factors. One key factor isthe proportional contribution of genetic material that helminthssurviving therapy will make to the next generation. Thiscontribution is influenced by the drug pressure (frequency andtiming of treatment), the drug efficacy, the gene flow (theintroduction of susceptible genotypes from elsewhere), thegeneration time and fecundity of the worms, the frequency ofresistance alleles prior to drug use, the number of genesinvolved, and the dominance or recessiveness of these genes.

Since it is quite difficult to set up experiments to examine theinfluence of these different factors, several mathematicalmodels have been developed to simulate the development ofAR in GI helminths (Barnes et al., 1995; Gettinby et al., 1990;Smith, 1990; Smith et al., 1999). Although, these models havetheir limitations and must certainly be interpreted with caution(Dobson, 1999), models such as the one of Barnes et al. (1995)concerning T. colubriformis in grazing sheep provides interest-ing insights. The model allowed up to three genes for drugresistance, each with two alleles, which were combinedindependently under random mating. Worms of all genotypeswere assumed to be equally fit in the absence of anthelmintic.The initial frequency of resistance alleles in the wormpopulation was assumed to be very low and was set at 0.01%.To examine the effect of using either mixtures of two drugs orrotations of a single one, two independent genes for resistanceto two drugs (with different mechanisms of action) weresimulated, with resistance being codominant and each drugkilling 99, 50, and 10% of worms of homozygous susceptible(SS), heterozygous (RS), and homozygous resistant (RR)genotypes, respectively. The simulations were run for a periodof 20 years, with treatment once a year for the ewes and threetimes a year for the lambs. These resulted in little developmentof resistance when the two drugs were used together (mixture).Substantial resistance, however, developed for all rotationstrategies, 1, 5, and 10 yearly, with slowest development of AR

in the annual rotation strategy. Assuming equal initial drugefficacy and equal resistance allele frequency, resistancedeveloped more rapidly if it was determined by a single genethan when two or more genes were involved. Furthermore,resistance evolved fastest when it was dominant, slower when itwas codominant and slowest when it was recessive. When 20%of the flock was never treated, resistance was delayed at theexpense of worm control. It should be noted, however, that thisand most other models are deterministic, ignoring the over-dispersed distribution of free-living and parasitic helminthstages.

Smith et al. (1999) used a stochastic model to examine theeffect of aggregated parasite distributions on parasite matingprobabilities and the spread and maintenance of rare (resistant)genotypes. They concluded that spatial heterogeneity intransmission might be a significant force in promoting thespread of resistant genotypes, at least when infection levels arelow. When modeling exercises are compared with currentknowledge of genetics of AR in helminths of livestock, the moststriking and alarming observation is the high frequencies ofresistance alleles observed in untreated populations of livestockhelminths of veterinary importance. Beech et al. (1994)analyzed individual genotypes of susceptible H. contortusbefore any exposure to BZ and reported initial frequencies ofresistance alleles of 46 and 12% at the isotype 1 and isotype 2 β-tubulin loci, respectively.

The available information, mainly on H. contortus, has beensummarized by Anderson et al. (1998). BZ resistance in thisparasite seems to be polygenic. At least two, possibly threegenes with recessive alleles are involved. LEV resistance in H.contortus and T. colubriformis is probably due to one singlemajor gene or gene cluster, the alleles of which are autosomalrecessive for the former and sex-linked recessive for the latter(Anderson et al., 1998). Resistance to IVM in H. contortusappears to be mediated by a single gene or gene complex withprimarily dominant effects. IVM resistance might thus developquite fast, as appears to be confirmed by field observations inSouth Africa, where IVM resistance in H. contortus developedafter only three treatments (van Wyk et al., 1989). Avermectinand mylbemycin resistance is now widespread in H. contortusand T. circumcincta of small ruminants all over the world butremarkably not in T. colubriformis (Sangster, 1999).

Detection of resistance

A wide range of tests has been developed to detect AR forresearch and diagnostic purposes (Presidente, 1985). Thegrowing importance of AR has led to an increased need forreliable and standardized detection methods (Coles et al., 1992)some of which have been previously described and reviewed(Presidente, 1985; Johansen, 1989; Hazelby et al., 1994; Tayloret al., 2002). Most of the methods described have drawbackseither in terms of cost, applicability and interpretation orreproducibility of findings (Várady and Čorba, 1999). The mostwidely used method for detecting and monitoring the presenceof AR in nematodes is the faecal egg count reduction test(FECRT), which is suitable for all types of anthelmintics

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including those that undergo metabolism in the host. Inaddition, a number of in vitro assays that measure the effectsof anthelmintics on development, growth or movement ofnematode stages have been developed as alternative methods ofdetection. These assays have been a useful tool for research butwith limited application for routine testing of isolates fromfarms especially where mixed populations of parasites arepresent. It is expected that anthelmintic chemotherapy willremain the mainstay of helminth control programmes in future.It is, therefore, vital that the efficacy of currently availablebroad-spectrum anthelmintics is maintained and that appropri-ate and reliable methods of monitoring the effectiveness of thesecompounds in the field are developed. Biochemical and/ormolecular studies of mode of action of different anthelminticswill pave the way to determine targets/enzymes responsible fordrug resistance which in turn would form the bases ofdiagnostic assays for AR.

In vivo tests

Faecal egg count reduction testThe most commonly used test to detect the problem of AR is

the FECRT, which compares the egg count before and aftertreatment with an anthelmintic drug (Boersema, 1983; Pre-sidente, 1985). A standardized protocol for the FECRT isavailable for the detection of AR in nematodes of veterinaryimportance (Coles et al., 1992). Generally, an untreated group isalso included to monitor any changes that occur in nematodeegg counts during the test period. One of the importantlimitations of FECRT is that test results may not estimateanthelmintic efficacy accurately because nematode egg outputdoes not always correlate well with actual worm numbers, andthe test only measures effects on egg production by matureworms. A good correlation has been found between faecal eggcounts and worm counts for H. contortus, but not for T.colubriformis (Sangster et al., 1979) or T. circumcincta (Martinet al., 1985). Egg counts for Nematodirus spp. are generally lowand bear little relationship to actual worm burdens (Chalmers,1985; Martin et al., 1985).

Faecal egg counts are generally high in goats but they do notcorrelate well with worm counts. If the interval between treat-ments is less than 10 days, egg production may be suppressedleading to an overestimation of anthelmintic efficacy with theBZ anthelmintics (Hotson et al., 1970; Martin et al., 1985). Forthis reason, the recommendation is to collect faecal samples 10–14 days after treatment (Coles et al., 1992). It has been observedthat FECRT can take the worker to a false situation either falsenegative (Jackson, 1993) or false positive (Grimshaw et al.,1996) due to different developmental stages of the parasite.

Egg counts are logarithmically transformed to stabilizevariances and expressed as geometric means for the groups(Sangster et al., 1979; Martin et al., 1982). Dash et al. (1988)argued that the arithmetic mean may be more appropriate inFECRTs because the geometric mean underestimates total eggoutput and transformation of data may vary between laborato-ries, thus, making comparisons difficult. A modification of theFECRT has been described in which no pre-treatment samples

are taken (Vizard and Wallace, 1987). The FECRT may notprovide sufficient information on its own for correctinterpretation.

Larval culture can be used to determine the species involved,but culture conditions may favour the development of onespecies over another (Presidente, 1985). Parasites with a highbiotic potential, e.g. H. contortus, may exert a disproportionateinfluence on the results and, therefore, correction factors have tobe included (Webb et al., 1979). It also appears that the testlacks the sensitivity to detect levels of resistance below 25%(Martin et al., 1989). Consequently, the use of maximumlikelihood mathematical techniques with a negative binomialstatistical model would aid in the early detection of AR usingfaecal egg count reductions and result in a lower probability ofinappropriately assigning an anthelmintic as effective. Recently,Torgerson et al. (2005) has demonstrated that negative binomialdistribution, which is a mathematical distribution model, candetect evidence of AR with a FECRT that otherwise mightrequire a slaughter trial to demonstrate. In addition, thesimulated data sets confirm that there is a significant probabilityof failure to detect low anthelmintic efficacy with commonlyused mathematical techniques.

On farm monitoring for nematode egg counts has becomepossible with the FECPAK system which was developed inNew Zealand (Coles, 2003). Once the investment in themicroscope-based kit i.e., FECPAK has been made, farmerscan determine both whether animals require treatment and if theanthelmintic is still effective. However, a simple definition ofresistance based on faecal egg counts is probably no longerpossible (Coles, 2005).

Critical anthelmintic testThe test is based on the collection of faeces from animals for

at least 4 days after anthelmintic treatment, from which theexpelled worms are recovered and their number estimated. Theanimals are slaughtered, residual worm burdens and thepercentage efficacy calculated by dividing the number ofexpelled worms by the residual number and multiplying by 100(Gordon, 1950). The major advantage of this test is that eachanimal serves as its own control and thus fewer animals arerequired than for the controlled efficacy test. However, it isunsatisfactory for estimating anthelmintic efficacy againstabomasal parasites of sheep because they undergo digestionduring their passage through the gut (Reinecke et al., 1962) andalso it is time and labor consuming (Johansen, 1989).

The controlled anthelmintic efficacy testThis test compares the worm burdens of animals post-

treatment which have been artificially infected with susceptibleor suspected resistant isolates of nematodes. The test can also beused to determine the anthelmintic activity against all stages ofdevelopment of parasites by slaughtering at varying times afterinfection (Reinecke et al., 1962). It is considered to be the mostreliable method for assessing AR and has, therefore, beenwidely used to confirm the results of FECRTs as well as forvalidating different in vitro tests (Boersema, 1983; Presidente,1985). However, it is the most costly test in terms of labour

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requirements and animal usage (Boersema, 1983) and is nowrarely used. In an attempt to reduce the costs and time taken,laboratory animal models have been used (Kelly et al., 1982).Guidelines for evaluating anthelmintic efficacy using thecontrolled test have been published previously (Clark andTurton, 1973; Powers et al., 1982; Wood et al., 1995).

Additional requirements for testing anthelmintics againstresistant strains were provided by Prichard et al. (1980) andColes et al. (1992). To characterize the sensitivity of a fieldisolate, groups of worm free animals can be inoculated withinfective larvae and the anthelmintic tested at 0.5, 1 and 2 timesthe recommended dose rate (Presidente, 1985). Inclusion in thetest of a known susceptible strain has been recommended(Prichard et al., 1980; Martin et al., 1982). With mixed wormpopulations, culturing needs to take account of the differentialdevelopment of certain species (Presidente, 1985). Resistance isgenerally confirmed when the reduction in geometric meanworm counts is less than 90%, or greater than 1000 wormssurvive treatment (Presidente, 1985).

In vitro tests

Avariety of different laboratory tests have been described forthe detection of AR in livestock helminths (Conder andCampbell, 1995). Those, which are most commonly used andwhich might be applied to detect AR in helminths are brieflydescribed here.

Egg hatch test

The egg hatch test (EHT) is an in vitro test, which is used onlyfor the detection of BZ resistance in livestock helminths. It isbased on the ovicidal activity of this group of molecules. An invitro EHT has been also described by Dobson et al. (1986) fordetecting resistance of nematodes to LEV. The original test wasdescribed by Le Jambre (1976). A standardized protocol wasadopted by the World Association for the Advancement ofParasitology (WAAVP) (Coles et al., 1992). Freshly collectedfaecal samples (within 3 h of being shed) are needed to obtainreliable data. If this is not possible, samples must be storedanaerobically. This storage does not influence the outcome of thetest at least for the major GI helminths of small ruminants (Huntand Taylor, 1989). This in vitro test has the advantage ofrequiring only one faecal sample. However, some authors havereported poor correlations between the results of the FECRTandthe EHT for helminths of livestock (Boersema et al., 1987;Dorny et al., 1994).

Unfortunately, the FECRT and the EHT is supposed to detectresistance only when at least 25% of the worm populationcarries resistance genes as shown by artificial infection ofanimals with mixtures of helminth populations with a knownlevel of AR (Martin et al., 1989). Since reversion tosusceptibility is considered to be possible only as long asresistance genes are present in less than 5% of the helminthpopulation (Roos et al., 1995), FECRT and EHT allow thedetection of AR only when it is too late to interfere. Field andexperimental data for helminths of livestock indicate that

reversion to susceptibility to anthelmintic drugs in livestockhelminths rarely occurs once resistance has been confirmed(Conder and Campbell, 1995).

The requirement for undeveloped eggs in EHT (Coles andSimpkin, 1977) has been amajor obstacle to the application of theEHT in routine diagnosis. As development proceeds beyond theventral indentation stage, a false positive result may be obtainedbecause sensitivity to thiabendazole decreases as embryonationproceeds (Le Jambre, 1976; Weston et al., 1984; Riou et al.,2005). A number of techniques have been described to avoid theproblems of screening for BZ resistance in the field. Whitlock etal. (1980) described the method to perform EHT in the field butdue to certain problems, various methods have been devised tostore eggs. Smith-Buijs and Borgsteede (1986) described thatfaecal samples can be stored at 4 °C for up to 3 days whilePresidente (1985) gave the idea of storing faecal samples in sealedpolythene bags with excluded air. Later on, Hunt and Taylor(1989) described about the anaerobic storage system of nematodeeggs. In spite of the above mentioned problems associated withEHT, it is still widely used to determine AR along with FECRT.

Larval development assay

The larval development assay (LDA) is more laborious andtime-consuming than the EHT but allows the detection ofresistance to the major broad-spectrum anthelmintic classes,including macrocyclic lactones. It was originally described byColes et al. (1988) and further improved by others (Gill et al.,1995; Hubert and Kerboeuf, 1992). In LDA, nematode eggs orL1 larvae are exposed to different concentrations of anthelmin-tics incorporated into agar wells in a microtiter plate or in a smalltest tube containing nutrient medium. The effect of the drugs onthe subsequent development into L3 larvae is measured. Theresults correlate well with those of in vivo tests. It is claimed thatthis test is more sensitive than FECRT and EHT and detects ARwhen about 10% of the worm population carries resistance genes(Dobson et al., 1996), but this remains to be proven why ithappens. It was concluded that the test could be run with anyanthelmintic to which resistance was suspected but it has beenreported to be reliable in detecting both the BZ and LEVresistance and IVM (Hubert and Kerboeuf, 1992). Nevertheless,Amarante et al. (1997) reported that this test appeared to beuseful in detecting resistance to BZ and LEV anthelmintics butrequired the use of an avermectin analogue other than IVM fordetecting resistance to the avermectin compounds.

Adult development test

In vitro techniques for the culture of trichostrongylidnematodes have been reported. For example, H. contortus hasbeen cultured through to the adult, egg laying stage (String-fellow, 1984, 1986). In vitro adult developmental assays fordetecting BZ resistance in H. contortus, based on these in vitroculture techniques have been described (Stringfellow, 1988;Small and Coles, 1993). However, there has been little furtherprogress in this area due to the complexity of the culturetechniques required.

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Larval paralysis test

A larval paralysis test was developed for the detection of LEVand morantel resistance (Martin and Le Jambre, 1979). In theassay, infective third stage larvae are incubated for 24 h in serialdilutions of the anthelmintic. After this time, the percentage ofparalysed larvae is determined at each concentration and a dose–response line plotted and compared to known reference strains.Boersema (1983) discussed the failure of this method to obtainrepeatable results and suggested the reversibility of paralysis as apossible cause. Geerts et al. (1989), however, reported fairly goodreproducibility of the test, any differences in repeatability beingattributed to the age of larvae. Sutherland and Lee (1990)described a modification of the larval paralysis assay byincubating third stage larvae of trichostrongyle nematodes ineserine, an acetylcholinesterase (AChE) inhibitor, and foundsuitable for detecting thiabendazole resistance due to the presenceof higher levels of AChE in resistant isolates.

Larval motility test

In vitro assays to detect resistance to BZ, macrocyclic lactonesor LEVand morantel have been described which are based on themotility of larvae (Conder and Campbell, 1995). For the lattergroup of anthelmintics, a clear cut distinction between susceptibleand resistant strains is not always possible (Geerts et al., 1989;Várady and Čorba, 1999). A similar motility test has been used toevaluate the sensitivity of Onchocerca volvulus microfilariae toIVM (Townson et al., 1994). Inmost of the in vitro assays, the endpoints are read microscopically and manually; consequently,assessments for biological activity have usually been qualitative,laborious, time consuming and highly variable. To render theinterpretation more objectively, a micromotility meter (Micro-motility Meter™, Band Instruments, 324, West south Street,Mason, MI 48854, USA) has been developed which enables toquantitatively assess drug effects on target helminthes in an invitro assay system. The test is claimed to be more sensitive,accurate, rapid, repeatable and inexpensive as compared to otherin vitro tests. A number of drugs can be used for the detection ofAR (Bennett and Pax, 1986; Folz et al., 1987). Folz et al. (1987)used this apparatus to detect drug resistance inH. contortus and T.colubriformis but Várady and Čorba (1999) found this test lessreliable, thus, this test has not been accepted wisely for thedetection of anthelmintic resistance.

Adult migration inhibition test

Amigration assay has been described, using adult stages of thepig nematode, Oesophagostomum dentatum, to differentiatebetween BZ and pyrantel susceptible and resistant strains. In theassay, adult worms removed on post-mortem were incubated inserial concentrations of anthelmintic for 30 min before transfer tothemigration chambers. Thesewere equippedwith polyamide netsof mesh size 300–500μm throughwhich theworms are allowed tomigrate over a period of 30 min. A dose–response curve was thenplotted based on the inhibition of migration by the mesh at thevarious drug concentrations used (Petersen et al., 1997, 2000). This

test can also be used in other species of nematodes of livestockincluding small ruminants but it has a limitation that animals haveto be sacrificed for the collection of adult worms.

Colorimetric assays

Biochemical assays, comparing non-specific esterases andacetylcholinesterases of BZ-resistant and -susceptible trichos-trongylid nematode strains have been described (Sutherland etal., 1988; Sutherland and Lee, 1989).

The mechanism of BZ resistance appears to be associatedwith a reduced affinity of tubulin for the anthelmintic (Sangsteret al., 1985; Lacey, 1985; Lacey and Prichard, 1986; Lacey etal., 1987). Based on these studies, Lacey and Snowdon (1988)described a diagnostic assay for the detection of BZ-resistantnematodes using the binding of tritiated BZ or carbamates totubulin extracts of third stage larvae.

Colorimetric assay is a modified version of the aphid tile test,which is used to detect insecticide resistance in single adult aphidsthat are resistant or susceptible to organophosphate or carbamateinsecticides. It is used to compare the levels of non-specificesterase in strains of the trichostrongyle nematodes which areknown to be resistant or susceptible to BZ anthelmintics.

Colorimetric assay has shown that there is significantly morespecific esterase in the infective-stage larvae of BZ-resistantstrains than in susceptible strains and this may prove to be of usein the detection of resistance to BZ anthelmintics (Sutherlandand Lee, 1989). This test has certain advantages that it isvaluable when fresh and unembryonated eggs are not availablefor use in EHT, where confirmation of a positive EHT, using adifferent method is required. It has also some limitations e.g., itrequires standard strain of parasite for comparison of colorchange and ambient temperature may affect the activity ofenzymes and hence change in color (Sutherland and Lee, 1989).

Polymerase chain reaction (PCR)

The molecular basis of specific AR is only known for theBZs where it is caused by a mutation in β-tubulin (Kwa et al.,1994). This information can be used to develop tests and studythe influence of management on the development of resistance(Humbert et al., 2001), however, keeping in mind that otherchanges may also contribute to BZ resistance, specificallytransport of the drug out of the parasite. Tests based on the useof a single point mutation to detect AR suffer from the potentialproblem that resistance may have resulted from more than onemutation. It has been assumed that BZ resistance is associatedwith a change from phenylalanine to tyrosine at position 200 inthe β-tubulin, but it is now becoming apparent that this does notalways hold true (Prichard, 2001). This requires the use of morethan one probe to ensure that resistance is detected.

The first specific primers to detect drug-resistant parasiticnematodes were developed by Kwa et al. (1994). These primersdiscriminated between heterozygous and homozygous BZ-resistantH. contortus for the alleles in question (β-tubulin isotype1), even when these genotypes are phenotypically indistinguish-able, and could also identify BZ-resistant T. colubriformis.

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According to Roos et al. (1995), PCR detected 1% of resistantindividuals within a susceptible worm population, a tremendousimprovement over other in vivo and in vitro tests. Elard et al.(1999) developed a more simplified method for the diagnosis ofBZ-resistant T. circumcincta. Using four primers (two allele-specific and two non-allele-specific ones) in the same PCR, adultworms were characterized for the mutation of residue 200 ofisotype 1β-tubulin. The technique has now been refined for use ona single worm, egg, or larva (Roos et al., 1995). Since thefrequencies of alleles associated with anthelmintic drug resistancemight be quite high even in susceptible populations, it is indeedimportant to examine DNA from individual parasite. If DNA isprepared from pooled parasites, the association between particularalleles is likely to be obscured (Anderson et al., 1998).Nevertheless, the examination of individuals may be time andcost consuming for getting a good appreciation of a parasitepopulation.

Since the same mutation is responsible for BZ resistance inmany parasitic nematodes, this method may provide a means ofinvestigating the frequencies of alleles bearing it in a wide rangeof animal and human intestinal nematodes.

Another interesting development is the availability of a P-gpgene probe for O. volvulus (Kwa et al., 1998). Since it has beenshown that P-gp plays a role in resistance to BZ and IVM in H.contortus (Blackhall et al., 1998; Xu et al., 1998; Kerboeuf et al.,1999, 2003), it can be expected that the same resistancemechanism might develop in many other helminths, includingO. volvulus.

Following use of the PCR, DNA is usually separated on agarelectrophoresis and the bands detected and recorded (Elard etal., 1999). If the ratio of ss, sr and rr individuals is required, thisremains the best test. However, a much more rapid test is the useof real time PCR or pyrosequencing that will determine the ratioof s to r genes in a population of worms. If probes for thespeciation of the nematodes were available, the ratio of speciesinvolved in resistance could also be determined at the egg stage.Real time PCR requires the use of very expensive equipmentbut saving of time may make up for the additional cost of thereagents and costs for the machine. It is probably too late to usereal time PCR for detecting BZ resistance in sheep and goats inmany parts of the world because it is so common.

The major difficulty with the use of real time PCR is that themolecular basis of LEV and macrocyclic lactone resistance isnot known and need not necessarily involve a single pointmutation (Coles, 2005). Further research is needed to knowexact mechanism of development of LEV and IVM resistance.

Control of resistance

Environmentally and/or immunologically based parasitecontrol strategies which seek to limit host/parasite contacthave an obvious application in the avoidance and managementof AR along with chemotherapy. Work to overcome AR hasbeen going on with increased intensity for more than a decade.The reason for this interest is multi-facetted but primarily drivenby the serious development of AR in parasite populations. Thefact that very few producers routinely screen for AR, coupled

with the relatively poor sensitivity of the most commonly usedin vivo screening methods, shows that most cases of resistanceare not detected at an early stage. This reduces the likelihood ofreversion occurring and thus, to avoid increasing levels ofresistance and/or the numbers of resistant species. The problemof AR can be circumvented either by delaying its onset or use ofalternate strategies in the form of integrated parasitemanagement.

Delaying the onset of anthelmintic resistance

Anthelmintic unexposed population of parasitesThe importance of keeping some nematodes in refugia

(unexposed to anthelmintic) was first time recognised byMichel (1985). Recently, van Wyk (2001) has provided animpetus to this concept and its importance has been given thefull recognition it deserves. The principle in insect resistancemanagement is that some areas are left untreated so that theseinsects provide the next generation. A similar principle appliesto nematodes. The contribution that worms surviving treat-ment make to the next generation must be limited. Thepractical implications of this policy have been discussed byColes (2002).

Researchers in South Africa (van Wyk et al., 1997) havedeveloped a very practical and promising technology, FAMA-CHA. This is a color score chart, which shows the eye mucosacolor at 5 different stages of anaemia due to infection with H.contortus. Based upon the degree of anaemia, recommendationsare made on when to treat. The chart has been developed forsheep and recently been adapted for goats. It is also possible, onthe basis of the readings, to make decisions about which animalsto use for further breeding and which to cull from the flock. Thistechnology is only meant for haematophagous worms andanybody who uses this chart should pay attention on bothparasitic and other diseases that might confound (causinganaemia) the readings. No system currently exists for decidingwhich animal infected with other non-haematophagous nema-todes can be left untreated.

Modelling of anthelmintic useModelling has shown that use of combination drenches

should reduce the chances of the development of resistance(Barnes et al., 1995), but combinations are more expensive thansingle drenches and some registration authorities have shown areluctance to allow their registration without extensiveadditional safety studies. In practice, mixtures have usuallybeen introduced once resistance is already present to one of themixtures so their full benefit has not been experienced.

Adoption of strict quarantine measuresFew farmers make a serious attempt to quarantine animals,

i.e., ensuring that resistant nematodes are not brought on to thefarm. If there is any doubt on the resistance status of nematodesin purchased sheep they should be treated with IVM and LEV,maintained in a yard for 48 h and then turned out onto conta-minated pasture (Dobson et al., 2001). This will ensure that anynematodes surviving treatment are diluted out by the nematodes

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already on the farm. However, the effectiveness of the strategyof quarantining has yet to be evaluated (Coles, 2005).

By reducing reliance on the use of anthelmintics, alternativemethods of nematode control should delay the development ofAR. In practice, apart from success in selecting for resistance tonematodes, particularly in New Zealand, most non-chemicalmethods of control have yet to be applied in the field (Coles,2005). To preserve the efficacy of remaining anthelmintics, itmay no longer be feasible, or desirable, to aim for completeparasite suppression. The most effective long-term strategiesmay seek to minimise the impact of parasites on sheep, whileaccepting that a degree of production loss is preferable to theexcessive exposure of worm populations to anthelmintics, withthe inevitable increase in levels of resistance.

Alternate strategies

Resistant animal breedsThe potential to breed sheep able to resist or tolerate parasitic

infections, with a consequent lower reliance on chemicaltreatments, has been recognised for some time. In Australia,breeding for resistance, which was found to have a greaterheritability of faecal worm egg count than resilience (Eady etal., 1996), has been promoted to sheep breeders, in particular, asthe ‘Nemesis’ extension programme. In addition to the benefitsof lower worm burdens, the reduced worm egg production byworm resistant sheep will have a favourable epidemiologicaleffect by decreasing the infectivity of pastures by worm larvae.However, breeding for animals with a higher resistance toparasitic GI nematodes has reached some level of success, but isfar from widely implemented since often such improvement hassome trade off with respect to productivity (Kloosterman et al.,1992; Woolaston and Baker, 1996; Gray, 1997).

Grazing management and anthelmintic treatmentsPlanned pasture movement systems can greatly reduce the

requirement for drenching while ensuring optimal nutrition foranimal performance. Strategies utilising ‘low worm’ pasturesinclude grazing crop stubbles, hay or bushfire aftermath, or alter-nate grazing with species such as cattle or horses, which do notshare major sheep parasites. However, optimising these strategieswill depend on knowledge of the ecology of worm larvae,especially an estimation of the time required for larval populationsto decline to safe levels after pasture contamination ceases. AsBarger (1999) pointed out, the behaviour of the free-livingparasitic stages is not well understood in many environments.

Treating then keeping on the same pasture for a while hasbeen recommended (Abbott et al., 2004) but will be difficultwhere persistent anthelmintics are used a some species will notrapidly reinfect the animals. A better way would be to movethen dose, but information on the level of infection of theanimals and on the new pasture would be required to knowwhen the optimal time to treat is (Molento et al., 2004).However, the heavy selection pressure placed on wormpopulations by movement to worm free situations aftertreatment has been recognised (Besier, 1996; Barger, 1999;van Wyk, 2001). In addition, lambs must also receive sufficient

exposure to parasites to develop an effective immunity againstinfection, while avoiding excessive larval intake. Grazingstrategies are primarily used as a tool to secure availability ofgrass (Thamsborg et al., 1999), but implementation of specificgrazing strategies resulted in alleviation of the impact of GINsin livestock (Barger, 1999). Research into the epidemiology ofworm infection in different environments, and the developmentof larval prediction systems, will greatly facilitate the adoptionof effective but sustainable grazing strategies for sheep wormcontrol.

Nutrition and parasite interactionThe pathophysiological consequences of GINs, including

inappetence, reduced efficiency of feed utilisation, and increasedendogenous loss of protein, is recognised as more severe inanimals on lower planes of nutrition (Coop and Holmes, 1996).The development of resilience against GINs could be a promisingconcept to dealwithAR. Protein supplementation can increase therate of acquisition of immunity and resistance to reinfection, andcan ameliorate the immune hypo-responsiveness of young sheep(Coop and Holmes, 1996). Significant benefits can be providedby feeding protein not degraded by rumen microbial activity (vanHoutert et al., 1995). Positive long-term effects of the short-termprovision of protein enriched diets on resistance to nematodes andproduction performance in sheep have been demonstrated (Dattaet al., 1999). There may also be a role for supplements based ontraceminerals, which in a deficient state may affect the expressionof immunity to helminths (Sykes and Coop, 2001). However,priorities for the allocation of limiting nutrients to immunologicalor other functions may vary with the physiological state of thehost, hence affecting the relative efficiency of supplementation atdifferent times (Coop and Kyriazakis, 1999).

Antiparasitic vaccinesA great deal of effort has been made to develop antiparasitic

vaccines by conventional approaches and, until recently, theonly vaccines to be successfully developed against helminthparasites in animals were irradiated larval vaccines againstDictyocaulus (D.) viviparus (Urquhart, 1985) and D. filaria(Sharma et al., 1981). In recent years, molecular biology hasbeen employed to develop new vaccines of interest to veterinaryparasitologists. The approach that is beginning to provesuccessful is to define a parasite antigen which confersresistance and to produce it by recombinant technology eitheras a polypeptide, by infecting the host with a benign expressionvector, or by vaccinating with a nucleic acid. Although, asubunit vaccine against Boophilus microplus has been devel-oped in Australia (Willadsen et al., 1995) but nothing effectivehas been found for nematodes. Vaccines against GINs basedupon naturally exposed or hidden antigens have beenthoroughly investigated (Smith, 1999; Dalton and Mulcahy,2001) over the last 10–15 years, but no commercial product hasbeen released on the markets based upon these results.

Botanical dewormersPlants have been used from ancient times to cure diseases of

man and animals. This system of therapy is commonly referred

2425A. Jabbar et al. / Life Sciences 79 (2006) 2413–2431

as ‘unani, folk, eastern, or indigenous’ medicine (Nadkarni,1954). The plant kingdom is known to provide a rich source ofbotanical anthelmintics, antibacterials and insecticides (Satya-vati et al., 1976; Lewis and Elvin Lewis, 1977). A number ofmedicinal plants have been used to treat parasitic infections inman and animals (Nadkarni, 1954; Chopra, 1956; Said, 1969).There are many plants which have been reported in the literaturefor their anthelmintic importance (Akhtar et al., 2000; Lateef etal., 2003; Iqbal et al., 2004, 2005, 2006a,b,c,in press-a,b; Jabbaret al., 2006, in press). Additionally, various tanniferous plantshave also been investigated for potential effect against eitherincoming parasite larvae and/or already established worms(Niezen et al., 1995, 1996, 1998). These plants can be apromising future for the control of worms which had previouslyshown resistance to synthetic drugs.

Biological controlOther possibility could be the use of nematophagous fungi to

control GINs but this concept has not yet been adopted by thefarmers. New technologies under development include the use ofnematophagous fungi to reduce larval worm populations onpasture, the potential anthelmintic activity of pasture speciescontaining condensed tannins or other active compounds. How-ever, even where successful, these approaches are unlikely toindividually provide the control obtained with the frequent useof effective anthelmintics. Hence, they are envisaged aselements of an integrated parasite management strategy, sothat an additive effect provides adequate worm control and long-term sustainability.

Recent studies have shown that deposited fresh faeces arequickly colonised by various species of these fungi (Hay et al.,1997; Bird et al., 1998). It has also been found that the fungi arepicked up by grazing livestock (cattle, sheep and horses) andsubsequently excreted in the voided faeces (Larsen et al., 1994;Manueli et al., 1999). These fungi belong to a taxonomicallydiverse group, with both egg parasitic, endoparasitic andpredacious nematode trapping fungi. This low degree ofsurvival subsequently impairs their ability to infect and kill asufficiently high number of developing parasite larvae in thedung environment. Within the group of nematode-trappingpredacious fungi several species have been tested against thefree-living infective stages of various ruminant parasiticnematodes. Fungi belonging to cattle and sheep have not beenstudied to a great extent due to the apparent lack of naturalability of the spores to survive in sufficient numbers through theGI tract of ruminants.

Conclusion

AR is a threatening problem to livestock industry posingvery threats to the future welfare and production of livestockthroughout the world. The factors considered most significanthave been an excessive frequency of treatments and theadministration of an inadequate dose (underdosing) particularlylatter is true for developing countries. However, these factorsmay not be completely true in all cases. Although some factorslike quality of drugs, education of farmers, modifications to

treatment practices also provide significant opportunities for thedevelopment of resistance. The exact mechanism of AR againstdifferent anthelmintics still needs to be elucidated which mayform the bases for easy and rapid diagnosis of AR in differentGINs against different anthelmintics. It may be concluded thatin future, sustainable control strategies for helminthosis mayrequire an integrated approach incorporating environmentalmanagement, and require chemoprophylaxis in order tominimize the pressure for parasite adaptation.

Unfortunately, such approaches are complex and climate-and parasite-specific and are thus not easily developed thanstrategies based on a single means of control. However, one ofthe main lessons to come out from studies on AR is that there isdire need of the time in intensive production systems to achieveacceptable standards of small ruminant health and welfare, thecosts of developing integrated approaches for control ofparasitism must be met.

Acknowledgements

This paper was prepared during the work on anthelminticresistance in the authors' laboratories which was supported by thePakistan Science Foundation and Higher Education Commissionof Pakistan.

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