Genetic variability following selection of Haemonchus contortus with anthelmintics

Review

29 Kleinschmidt, I. et al. (2000) A spatial statistical approach to malaria mapping. Int. J. Epidemiol. 29, 355–361 30 Connor, S.J. (1999) Malaria in Africa: the view from space. Biologist 46, 22–25 31 Thomson, M.C. et al. (1999) Predicting malaria infection in Gambian children from satellite data and knowledge of bednet usage: the importance of spatial correlation in the interpretation of the results. Am. J. Trop. Med. Hygiene 6, 2–8 32 Thomson, M.C. et al. (1994) Malaria prevalence is inversely related to vector density in The Gambia, West Africa. Trans. R. Soc. Trop. Med. Hygiene 88, 638–643 33 Hay, S.I. et al. (2000) Annual Plasmodium falciparum entomological inoculation rates (EIR) across Africa: literature survey, internet access and review. Trans. R. Soc. Trop. Med. Hygiene 94, 113–127 34 Snow, R.W. et al. (1997) Relation between severe malaria morbidity in children and level of Plasmodium falciparum transmission in Africa. Lancet 349, 1650–1654 35 Smith, T.A.et al. (2001) Child mortality and malaria transmission intensity in Africa. Trends Parasitol. 1, 3 36 Gupta, S. et al. (1999) Immunity to non-cerebral severe malaria is acquired after one or two infections. Nat. Med. 5, 340–343 37 Wilson, D.B. et al. (1950) A review of hyperendemic malaria. Trop. Dis. Bull. 47, 677–698 38 Coleman, P.G. et al. (1999) Rebound mortality and the cost-effectiveness of malaria control: potential impact of increased mortality in late childhood following the introduction of insecticide treated nets. Trop. Med. Int. Health 4, 175–186 39 Phillips-Howard, P.A. (1999) Epidemiological and

TRENDS in Parasitology Vol.17 No.9 September 2001

40

41

42

43

44 45

46

47

48

49

50

control issues related to malaria in pregnancy. Ann. Trop. Med. Parasitol. 93, S11–S17 Rogier, C. et al. (1999) Plasmodium falciparum clinical malaria:lessons from longitudinal studies in Senegal. Parassitologia 41, 255–260 Brabin, B.J. et al. (2001) An analysis of anemia and pregnancy-related maternal mortality. J. Nutr. 131, 604S–614S Brabin, B.J. et al. (1999) A birthweight normogram for Africa as a malaria control indicator. Ann. Trop. Med. Parasitol. 93, S43–S57 Verhoeff, F.H. et al. (1999) Increased prevalence of malaria in HIV-infected pregnant women and its implications for malaria control. Trop. Med. Int. Health 4, 5–12 Stephenson, L.S. et al. (2000) Global malnutrition. Parasitology 121, S5–S22 Rice, A.L. et al. (2000) Malnutrition as an underlying cause of childhood deaths associated with infectious diseases in developing countries. Bull. WHO 78, 1207–1221 Shankar, A.H., (2000), Nutritional modulation of malaria morbidity and mortality. J. Infect. Dis. 182, S37–S53 Shankar, A.H. et al. (2000) The influence of zinc supplementation on morbidity due to Plasmodium falciparum: a randomized trial in preschool children in Papua New Guinea. Am. J. Trop. Med. Hygiene 62, 663–669 Fontaine, R.E. et al. (1961) The 1958 malaria epidemic in Ethiopia. Am. J. Trop. Med. Hygiene 10, 795–803 Cox, J. et al. (1999) Mapping malaria risk in the highlands of Africa. MARA/HIMAL Technical Report Vaahtera, M. et al. (2000) Epidemiology and predictors of infant morbidity in rural Malawi.

445

Paediatr Perinatal Epidemiol. 14, 363–371 51 Pison, G. et al. (1993) Rapid decline in childmortality in a rural area of Senegal. Int. J. Epidemiol. 22, 72–80 52 Ashley-Koch, A. et al. (2000) Sickle hemoglobin (Hb S) allele and sickle cell disease: A HuGE review. Am. J. Epidemiol. 151, 839–845 53 Trape, J.F. et al. (1998) Impact of chloroquine resistance on malaria mortality. Comptes redus de l’Academie des Sciences Serie III - Science de la Vie. 321, 689–697 54 Onori, E. and Grab, B. (1980) Indicators for the forecasting of malaria epidemics. Bull. WHO 58, 91–98 55 Lindblade, K.A. et al. (2000) Early warning of malaria epidemics in African highlands using Anopheles (Diptera: Culicidae) indoor resting density. J. Med. Entomol. 37, 664–667 56 Thomson, M.C. et al. (2000) Environmental information systems for multi–Disease surveillance and epidemic prediction. In Geography and Medicine, GEOMED’99 (Flahault, A., et al. eds), pp.120–128, Elsevier 57 Hay, S.I. et al. (2000) Etiology of interepidemic periods of mosquito-borne disease. Proc. Natl. Acad. Sci. USA 97, 9335–9339 58 Nuttall, I. et al. (1998) Systems of geographic information and the campaign against tropical diseases. Med. Trop. 58, 221–227 59 Bernardi, M. (2001) Linkages between FAO agroclimatic data resources and the development of GIS models for the control of vector-borne diseases. Acta Tropica 79, 21–34 60 Thomson, M.C. et al. (1995) Entomological evaluation of The Gambia’s National Impregnated Bednet Programme. Ann. Trop. Med. Parasitol. 89, 229–241

Genetic variability following selection of Haemonchus contortus with anthelmintics Roger K. Prichard Genetic diversity in nematodes leads to variation in response to anthelmintics. Haemonchus contortus shows enormous genetic diversity, allowing anthelmintic resistance alleles to be rapidly selected. Anthelmintic resistance is now a widespread problem, especially in H. contortus. Here, I compare the genes involved in anthelmintic resistance in H. contortus with those that confer susceptibility or resistance on the free living nematode Caenorhabditis elegans. I also discuss the latest knowledge of genes associated with resistance to benzimidazoles, levamisole and the macrocyclic lactones and the need for DNA markers for anthelmintic resistance.

Parasites do not respond uniformly to treatment. A dose titration assay of a parasite population will almost always reveal variation in responses to an http://parasites.trends.com

anthelmintic until high efficacy is achieved. This is due to genetic diversity in the parasite population. Furthermore, treatment eliminates parasites whose genotype renders them susceptible, and the eliminated worms cannot pass on their ‘susceptibility’ alleles to offspring. Parasites that are resistant (Box 1) to the dose survive and can pass on their ‘resistance’ alleles. The frequency and intensity of treatment, and the extent of dilution of ‘resistance’ alleles by ‘susceptibility’ alleles in the reproducing population, by parasites that establish after the treatment, determine the rate of selection for resistance.

1471-4922/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1471-4922(01)01983-3

446

Review

TRENDS in Parasitology Vol.17 No.9 September 2001

Box 1. Anthelmintic resistance In nematodes of small ruminants, and especially in Haemonchus contortus, anthelmintic resistance threatens livestock production in parts of the worlda,b. Resistance to benzimidazoles (BZ), ivermectin (IVM) and other macrocyclic lactones (MLs), and to levamisole (LEV) and pyrantel, is also found in other trichostrongylid nematodesc,d. Resistance to all of the available anthelmintic classes, with the exception of the MLs, is also widespread in cyathostome nematodes of horsese. Perhaps surprisingly, IVM resistance has not been demonstrated in cyathostomes despite many years of intensive treatment of horses with IVM. Anthelmintic resistance does occur in cattle nematodes but the problem is not yet severe. There are reports of BZ and LEV resistance in Ostertagia ostertagi, Cooperia oncophera and Trichostrongylus axei in cattlef–k, and there have recently been reports of IVM resistance in Cooperia spp., Trichostrongylus spp.k–n and Haemonchus placei in cattle (F. Echevarria and A. Pinheiro, unpublished; F. Paiva and I. Menz, unpublished). So far, there have been no documented problems of anthelmintic resistance in gastrointestinal nematodes of humans, or in Onchocerca volvulus and lymphatic filaria. However, anthelmintic use is increasing and there is concern that resistance could become problematic in these human nematode parasiteso,p. There is no reason to believe that alleles that confer resistance are not present in some species of nematodes of humans and cattle. There are factors that might increase the proportion of a nematode population in refugia in the case of human and cattle parasites, which might mitigate against rapid selection for resistance. In addition, treatment frequencies have usually been greater in small ruminants and horses than in humans and cattle. However, anthelmintic use is increasing in people and cattle. Long-acting boluses, treatment of large human populations to break transmission of filarial nematodes and the tendency to increase the frequency of IVM treatment in

onchocerciasis from once per year to up to two or more times per year will increase selection pressure for resistance. References a Waller, P. (1997) Anthelmintic resistance. Vet. Parasitol. 72, 391–412 b Jackson, F. and Coop, R.L. (2000) The development of anthelmintic resistance in sheep nematodes. Parasitology 120 (Suppl), S95–S107 c Sangster, N.C. and Gill, J. (1999) Pharmacology of anthelmintic resistance. Parasitol. Today 15, 141–146 d Gopal, R.M. et al. (1999) Resistance of field isolates of Trichostrongylus colubriformis and Ostertagia circumcincta to ivermectin. Int. J. Parasitol. 29, 781–786 e Woods, T. et al. (1998) Anthelmintic resistance on horse farms in north central Florida. Equine Practice 20, 14–17 f Eagleson, J.S. and Bowie, J.Y. (1986) Oxfendazole resistance in Trichostrongylus axei in cattle in Australia. Vet. Res. 119, 604 g Geerts, S. et al. (1987) Suspected resistance of Ostertagia ostertagi to levamisole. Vet. Parasitol. 23, 77–82 h Jackson, R.A. et al. (1987) Isolation of oxfendazole resistant Cooperia oncophora in cattle. N. Z. Vet. J. 35, 187–189 i Eagleson, J.S. et al. (1992) Benzimidazole resistance in Trichostrongylus axei in Australia. Vet. Rec. 131, 317–318 j Borgsteede, F.H.M. et al. (1992) Studies on an Ostertagia ostertagi strain suspected to be resistant to benzimidazoles. Vet. Parasitol. 41, 85–92 k Vermunt, J.J. et al. (1995) Multiple resistance to ivermectin and oxfendazole in Cooperia species of cattle in New Zealand. Vet. Rec. 137, 43–44 l Vermunt, J.J. et al. (1996) Inefficacy of moxidectin and doramectin against ivermectin-resistant Cooperia spp. of cattle in New Zealand. N. Z. Vet. J. 44, 188–193 m Coles, G.C. et al. (1998) Ivermectin-resistant Cooperia species from calves on a farm in Somerset. Vet. Rec. 142, 255–256 n Fiel, C.A. et al. (2000) Resistance of trichostrongylid nematodes – Cooperia and Trichostrongylus – to avermectin treatments in cattle in humid pampa, Argentina. Rev. Med. Vet. (Buenos Aires) 81, 310–315 o Meredith, S.E.O. and Dull, H.B. (1998) Onchocerciasis: the first decade of Mectizan treatment. Parasitol. Today 14, 472–474 p WHO (1998) Report of the WHO Informal Consultation on Monitoring of Drug Efficacy in the Control of Schistosomiasis and Intestinal Nematodes, Geneva, 8 June – 1 July, World Health Organization

Genetic diversity in Haemonchus contortus and other nematodes

Roger K. Prichard Institute of Parasitology, McGill University, Montreal, Canada H9X 3V9. e-mail: [email protected]

The diversity observed in several nematodes, but not all of them1, is startlingly large. The genetic composition of an organism includes both the nuclear DNA and the mitochondrial DNA (mtDNA). The complete sequence of mtDNA is known for the nematodes Ascaris suum, Caenorhabditis elegans2 and Onchocerca volvulus3, and sequence information on a number of trichostrongylid nematodes has been compared4. Nematode mtDNA undergoes a high rate of mutation5 and the mutation rate in Haemonchus contortus is up to ten times higher than that of vertebrates4. The nuclear DNA of H. contortus is extremely diverse. Nuclear diversity in H. contortus has been studied in the genes encoding two β-tubulins6,7, several P-glycoproteins8,9 (Pgps), two glutamategated chloride channel subunits (GluCla and a GluCl β subunit), an N-acetylcholine receptor, a phosphoenolpyruvate carboxykinase10 and tandemrepeat-type galectins, as well as in a Tc1 transposable element11 and transposon integration and http://parasites.trends.com

microsatellite analyses12–14. All these genetic analyses indicated high diversity. Genetic diversity can be within a population or between populations (geographically separated or drug selected). Studies in four species of trichostrongylid nematodes indicated that 96–99% of nucleotide diversity is found within populations15. H. contortus shows great genetic diversity both within populations and between distinct isolates. Polymorphism depends on the mutation rate, the effective size of the population and migration rates. H. contortus is an extremely successful parasite, found in many ruminant species from the humid tropics to cool temperate climates. With the domestication of ruminants, its host population size is huge, and trade in livestock leads to migration of the parasite. Individual ruminants can harbor hundreds to thousands of H. contortus individuals. It is a prolific breeder, with one female worm producing up to 10 000 eggs per day. The population size on pasture is usually much greater than that within ruminants. The effective population size of H. contortus is huge. This population size, its high reproduction rate and

Review

TRENDS in Parasitology Vol.17 No.9 September 2001

the enormous range of its environment are conducive to extreme genetic diversity. In addition to genetic diversity, nematodes use alternative splicing of mRNA to produce different proteins from the same gene, as well as posttranslational modifications, to increase physiological diversity. For example, C. elegans can produce three different γ-aminobutyric acid (GABA) receptor subunit proteins by alternative splicing of the same gene16. Genes involved in anthelmintic action

Levamisole (LEV) is a nicotinic cholinergic agonist causing opening of non-selective cationic channels, which results in depolarization of nematode musclecell membranes. Benzimidazoles (BZs) bind to tubulin, leading to the disruption of microtubules. Mutations or deletions in genes encoding β-tubulins have been found to be associated with a change in phenotype from susceptibility to resistance in H. contortus17,18. The macrocyclic lactone (ML) anthelmintics paralyse the pharynx, the body wall and the uterine muscles of nematodes. In adult H. contortus, the pharynx appears to be the most sensitive of these muscles19. Early studies of the mechanism of action of avermectins (AVMs) showed that they are GABA-receptor antagonists, blocking hyperpolarization of nematode somatic muscle membranes20. Recently, a putative GABA-receptor subunit (HG1) has been localized along the ventral nerve cord and in several neurons in the head and possibly nerve ring in H. contortus21. If such a ligandgated receptor is involved in the action of MLs, it would be consistent with earlier work22 that indicated that paralysis of nematode body muscle by MLs resulted from a hyperpolarization of neurons, and thus from an inhibition of excitatory signals to the muscles rather than from a direct inhibition of body muscle cells. However, considerably higher concentrations of AVM are required for GABA-antagonist effects than for effects on the glutamate-gated chloride channels23 (GluCl) that are found uniquely in invertebrates. The GluCls were first cloned from C. elegans24. The gene encoding the GluCl α subunit (avr-15) is expressed in the pharynx of C. elegans25,26 and has IVM and glutamate receptors25. Two alternatively spliced GluCl subunit cDNAs (HcGluCla and HcGluClb) have been cloned from H. contortus27, and the longer sequence has been expressed and found to bind IVM with high affinity (Kd ≈ 10−10 M). Three other H. contortus GluCl subunit cDNAs have been sequenced: Hc-GBR2A and its alternatively spliced form Hc-GBR2B (Ref. 28), which have high homology to C. elegans avr-14, and HG4 (Ref. 29). HcGRB2 expression has been localized to the nerve ring, the ventral and dorsal nerve cords, the anterior portion of the dorsal sub-lateral cord, and motor-neuron commissures in H. contortus28, whereas HG4 has been localized to motor neuron commissures in the anterior portion of H. contortus from the nerve ring to just anterior of the vulva, including possible http://parasites.trends.com

447

nerve cord staining, but no expression on pharyngeal muscle was detected29. Different GluCl subunits might show variable sensitivity to MLs and different sites of expression, which could account for the paralytic effects of MLs on different neuromuscular systems at different ML concentrations and for the different sensitivities of the nematode life cycle stages; the level of expression of H. contortus GluCla was found to be adult > L3 > eggs27. It has been found that feeding is inhibited by MLs in adult H. contortus at about 10−9 M (Ref. 19) and in developing larvae (L1 and L2) by 10−8 M (Ref. 30), whereas migration of H. contortus L3 is inhibited by ML at 3 × 10−7 M (Ref. 31). In vivo, susceptible H. contortus are expelled 8–10 h after IVM treatment, suggesting that paralysis of body wall muscle might be critical for this rapid expulsion, even though paralysis of pharyngeal muscle is more sensitive. However, ML anthelmintics persist, at decreasing concentrations, for several days. As the ML concentration decreases, motility might be regained, but paralysis of the pharynx and inhibition of feeding might endure longer than body muscle paralysis and contribute to worm death. The accumulating evidence suggests that several genes are involved in the mechanism of action of the MLs. Associations between genes and anthelmintic resistance

Some methods for assessing genetic variation in parasitic nematodes have been reviewed previously32. However, a number of recent approaches are particularly relevant to the study of anthelmintic resistance. C. elegans has been used to study the effects of gene knockouts and mutations on resistance to anthelmintics. This approach has identified over 20 loci in this free-living nematode that can confer low-level ML resistance, and the deletion or mutation of additional loci can confer high-level ML resistance33. However, many of the resistant C. elegans mutants have mobility, egg laying, amphid and pharyngeal dysfunctions that would be lethal in a parasite and thus be irrelevant to resistance in parasites. Highlevel resistance in C. elegans is also not likely to be relevant to resistance in parasites. Gene knockouts and induced mutations cannot be readily used to study resistance genetics in parasites. However, C. elegans can help our understanding of genetic associations with resistance in parasitic nematodes in two ways: (1) it can help to identify possible candidate gene homologs in the parasites; and (2) it can be used to study the phenotype of parasitic nematode alleles in transgenic C. elegans. This approach has been used34 to show that the Phe200Tyr mutant of H. contortus isotype-I β-tubulin, when heterologously expressed in C. elegans, conferred BZ resistance. A common approach to studying associations between different loci and resistance has been to look for mutations in independent drug-sensitive and -resistant populations of parasites by restriction

448

Review

TRENDS in Parasitology Vol.17 No.9 September 2001

fragment length polymorphism (RFLP). Although this approach can indicate that different populations differ at a particular locus, it is not specific for anthelmintic resistance unless the resistant population is derived from the susceptible population. Differences in a loci between susceptible and resistant populations that are genetically independent might simply be a reflection of genetic isolation of the populations. However, when a resistant population has been derived from a susceptible population solely by drug treatment, the resistant and susceptible populations will share the same genetic background except for the effects of the drug selection. Such comparisons of resistant strains with parental susceptible strains have been successfully used7 to compare polymorphism in genes encoding isotype-I and isotype-II β-tubulins. Similar approaches, using RFLP and single strand conformational polymorphism, on IVM-selected, MOX-selected and parental unselected strains were used8,10 to elucidate genes associated with ML resistance in H. contortus. If the alleles for susceptibility and resistance are identified, they can be expressed and the products and their function and drug affinities compared. This approach can pinpoint mutations that confer resistance and provide information on what causes the resistance and how it can be accurately detected. Recently, it has been used successfully to show that BZ resistance in H. contortus can be due to mutations at two codons in both isotype-I and isotype-II β-tubulins (R. Prichard et al., unpublished). Another approach to identifying genes associated with anthelmintic resistance in H. contortus has been to produce hybrids by crossing anthelminticresistant H. contortus with susceptible Haemonchus placei35. Hybrid males are sterile but females can be backcrossed with H. placei to give a background of H. placei but with resistance-associated genes from the H. contortus ancestor. Once the backcrosses have been made, the anthelmintic-selected hybrids can be screened for any candidate gene to see whether that gene originated in H. contortus or H. placei by sequence analysis and the frequency of each type. This can be a definitive means of showing an association of a gene with resistance. Results must however, be interpreted with some care. The association does not necessarily mean that the gene that shows a significantly different frequency from that expected from random matings is itself involved in resistance. It might be located near a gene that is involved in resistance and be linked during segregation. Another caveat is that the sequence analyzed be always uniquely different between H. contortus and H. placei. In the analysis35, ~700 bp were sequenced. No polymorphism at all was described for H. placei in this fragment, even though the H. placei population was not exposed to drug selection. This is surprising given the high diversity of unselected H. contortus and other http://parasites.trends.com

nematodes. The anthelmintic resistant H. contortus showed two polymorphs for this fragment. This limited polymorphism for the H. contortus could be expected as the CAVRS strain of H. contortus used had been previously subjected to severe bottlenecking during the experimental ML selection protocol. On the basis of the polymorphism observed in the resistant H. contortus strain and the susceptible H. placei strain, a ClaI site that was apparently unique to the single H. placei polymorph and another BamHI site that mostly occurred in H. placei but was also present in the less frequent H. contortus allele were used to analyze the gene ancestry. Should the H. placei be more diverse than was thought in the fragment analysed and contain a low frequency of alleles without the ClaI site, these alleles would be counted as H. contortus rather than H. placei, leading to an overestimate of H. contortus alleles in the anthelmintic-selected backcross population. This could produce a misleading result. This method therefore requires a careful analysis of the polymorphism in the fragment being analyzed in each population. A third caveat is that, as with most of the methods discussed for analyzing anthelmintic resistance, the results are relevant only to the populations being investigated. Nevertheless, the hybridization technique can be a powerful tool for investigating genetic associations with anthelmintic resistance. Resistance-associated genes in H. contortus

Genes involved in the mechanism of resistance might (but will not necessarily) be involved in the mechanism of action of an anthelmintic. Resistance mechanisms can be due to changes in the drug receptor or to modulation of drug concentration. Genes that code for proteins involved in transport or metabolism can be involved in drug resistance but have no direct role in the mechanism of action. If the mechanism of resistance is due solely to mutation(s) in genes involved in the mode of action, anthelmintics with the same mode of action are likely to share the same mechanism of resistance. However, if the mechanism of resistance is due entirely or in part to modulation of drug concentration, different anthelmintics in the same mode of action class might not show the same level of resistance because of the effect of different chemical substituents on transport or metabolism. LEV-resistant mutants of C. elegans lack a normal complement of acetylcholine (ACH) receptors36. The LEV-resistance genes lev-1, unc-29 and unc-38 encode subunits of ACH-gated cation (Na+ and K+) channels in C. elegans, including a LEV–morantel binding site37. The lev-1 gene appears to be a structural subunit of a five-subunit ACH-gated channel. The Glu237Lys mutation in a transmembrane region of LEV-1 changes the channel from cationic to anionic and renders C. elegans insensitive to LEV. In Oesophogostomum dentatum, there appear to be a number of cholinergic channels that respond to LEV with variable conductances38. In H. contortus, there are high- and low-affinity LEV binding sites on

Review

TRENDS in Parasitology Vol.17 No.9 September 2001

(a) CH3 O

C

α-tubulin

N

167

N N

N

N

β-tubulin 200

(b)

C terminus N terminus

O

Cysteine 201

C

C C

S C

N O

C

C

N H H

N

C

Phenylalanine 200

O

C

N

O

C

C

O

Serine 166

N

N

C

C C

Benzimidazole

O

Phenylalanine 167

N

TRENDS in Parasitology

Fig. 1. Proposed benzimidazole (BZ) binding to tubulin. (a) The BZ-binding site involves α- and β-tubulins, and the phenylalanines at codons 167 and 200 of β-tubulin in Haemonchus contortus. The planar BZ ring system is proposed to bind between the phenyl rings of Phe167 and -200, and to form a covalent bond between Cys201 and the BZ carbamate. (b) Mutation of either Phe167 or Phe200 to phenolic Tyr prevents BZ from locking into Phe167 or Phe200 binding site.

cholinergic receptor subunits. The low-affinity receptors appear to be unresponsive to LEV in resistant worms39. These studies suggests that several genes are involved with the effects of LEV and with resistance to LEV. In one isolate of H. contortus, LEV resistance was multigenic40. Other work suggested that it was a recessive autosomal trait in this parasite41. Although ACH-gated cation channel subunit genes seem to be involved, further work needs to be done to determine the specific genes associated with LEV resistance in H. contortus. BZ resistance in H. contortus has been shown to be due to selection on genes for isotype-I (β8 and β9) and isotype-II ( β12–β16 ) β-tubulins6,7,42. A Phe200Tyr mutation of isotype-I β-tubulin is associated with the BZ resistance43. We have also shown, using pointdirected mutagenesis, expression and BZ binding and polymerization studies, that the Phe200Tyr mutation on isotype II also contributes to BZ resistance1 and that, in some BZ-resistant strains, a null isotype II can http://parasites.trends.com

449

be selected6. In addition to the Phe200Tyr mutation, we have found BZ-resistant populations with Phe at amino acid 200 but with a Phe167Tyr or Phe167His mutation, and subsequently shown that this codon-167 mutation can confer BZ resistance1. These findings [that both isotype-I and isotype-II β-tubulins are involved with BZ resistance and mutations at both codon 167 and codon 200 (Fig. 1) can confer resistance] have important implications for the design of genetic markers for BZ resistance. A DNA-based method, based on the Phe200Tyr mutation in isotype I, has been described for diagnosis of BZ resistance44. This might be predictive most of the time but might give false negatives. DNA methods for detecting BZ resistance should be based on changes at codons 167 and 200 of both isotype-I and isotype-II β-tubulins (or the null isotype II) to provide an accurate assessment of the BZ-resistance genotype in H. contortus (Box 2). GluCl genes are implicated in the action of MLs. Recent findings in C. elegans have led to a hypothesis for IVM action on the pharynx of this worm (Fig. 2), resulting in starvation, and for IVM resistance in C. elegans45. According to this hypothesis, products of the genes avr-15 (GluClα2), avr-14 (GluClα3) and GLC-1 (GluClα1) might respond to IVM. The avr-15 gene is expressed in pharyngeal muscle, allowing IVM to act directly on the pharynx. By contrast, avr-14 and GLC-1 are expressed on extrapharyngeal neurons, which are connected to the pharyngeal cells via linking neurons in which the gap junction innexins (produced by the genes unc-7 and unc-9, which do not bind IVM themselves), convey the hyperpolarization signal from the extrapharyngeal neurons to the pharynx. In this model45, the amphid dye filing Dyf genes, such as osm-1, might act additively to regulate IVM uptake and access to the various GluCl and IVM receptors. Different AVM-resistant strains of H. contortus show different phenotypes31 and it has been argued that these differences might reflect selection pressures46. These findings suggest that, as in C. elegans, more than one gene might contribute to ML resistance in H. contortus. Selection pressure might well influence the role of different genes in ML resistance. However, other factors (such as the genetic diversity of different populations and the ML used in the selection) might also influence which genes respond to ML selection in different populations. Different ML-resistant populations might not show identical genetic responses. Crossing the IVM-resistant CAVRS strain of H. contortus with a susceptible strain and then assessing phenotype with the larval development assay has shown that resistance in CAVRS is completely dominant and mainly under the control of a major gene. However, in the larvae, this gene mapped to an autosomal loci, whereas in adult H. contortus expression of resistance was sex linked, being stronger in females than males47. These differences between the genetics of resistance in larvae and adults suggest that resistance might

450

Review

TRENDS in Parasitology Vol.17 No.9 September 2001

Box 2. β-Tubulin genes involved in benzimidazole resistance Two β-tubulin loci are involved in benzimidazole (BZ) susceptibility and resistance, making five (six if the isotype-II null is considered separately) different genotypes (Table I). The relationship between genotype and phenotype for two different sets of observations on trichostrongylid nematodes can be calculated. The frequency of an RFLP genotype (1A) of isotype-I β-tubulin that was associated with BZ resistance was founda to be 0.458 in a susceptible strain, increasing to 0.975 in a BZ-resistant strain that was selected from the susceptible strain, and to be 0.933 in another BZ-resistant field strain. At isotype-II β-tubulin, the estimated frequency of a BZ-resistance genotype (2A) was 0.090 in the susceptible strain, increasing to 0.694 and 0.680 in the two BZ-resistant strains, respectively. One can calculate that the frequency of homozygous resistant alleles at both isotype I and isotype II (1A/1A plus 2A/2A) will be (0.458)2 × (0.090)2 = 0.0017. That is, ~0.2% of the susceptible Haemonchus contortus strain will be homozygous resistant at both loci, whereas ~ (0.975)2 × (0.694)2 = 0.458 (45.8%) of the BZ-resistant strain will be homozygous resistant for BZ at both loci. These calculations are similar to the experience from treatment of BZ-susceptible and -resistant populations, in which efficacy results of ~99.8% and ~50% for susceptible and moderately resistant isolates of H. contortus, respectively, are common.

Further selection with BZ could increase the frequency of the resistance allele at isotype II or select for the null isotype II. This model is consistent with incompletely recessive BZ resistance involving more than one gene, as has been found in genetic studies in H. contortus b,c. It is interesting that similar frequencies were foundd for BZ-susceptible and resistant alleles at isotype-I β-tubulin in a susceptible isolate of Trichostrongylus colubriformis, as was found in H. contortusa. In T. colubriformis, the frequency of homozygous resistance alleles for isotype I in the susceptible population was 5.8%. This would seem to be extremely high for a susceptible population if only a single loci was involved in BZ resistance and it seems likely that, in T. colubriformis as in H. contortus, both isotypes I and II β-tubulins contribute to BZ resistance. References a Beech, R.N. et al. (1994) Genetic variability of the β-tubulin genes in benzimidazole-susceptible and -resistant strains of Haemonchus contortus. Genetics 138, 103–110 b Sangster, N.C. et al. (1998) Inheritance of levamisole and benzimidazole resistance in an isolate of Haemonchus contortus. Int. J. Parasitol. 28, 503–510 c Le Jambre, L.F. et al. (1979) The inheritance of thiabendazole resistance in Haemonchus contortus. Parasitology 78, 107–119 d Grant, W.N. (1994) Genetic variation in parasitic nematodes and its implications. Int. J. Parasitol. 24, 821–830

Table I. Genotypes involved in benzimidazole resistance in Haemonchus contortus BZ resistance genotypea,b Notes

Efficacy at recommended dose rates

Most susceptible S1/S1, S2/S2

Nematodes susceptible to low concentrations of BZs and less potent BZs, such as thiabendazole

~100%

High susceptibility to potent BZs such as albendazole, fenbendazole and oxfendazole, but resistance evident with less potent BZs

90–98%

Very resistant to less potent BZs, reduced efficacy even with potent BZs

20–90%

Haemonchus contortus with these β-tubulin alleles show strong resistance to all BZs

~0%

Slightly resistant S1/R1, S2/S2

Moderately resistant R1/R1, S2/S2 R1/R1, S2/R2 Highly resistant R1/R1, R2/R2 R1/R1, Ø2/Ø2 aBenzimidazoles

(BZs) work by inhibiting tubulin polymerization and promoting shortening of microtubules. and S2 are BZ-susceptibility alleles for β-tubulin loci in isotypes I and II, respectively. R1 and R2 are β-tubulin alleles for BZ resistance in isotypes I and II, respectively. Ø2 represents a null isotype II. bS 1

involve more than a single gene. The CAVRS resistant strain was experimentally selected in which the progeny of the very few survivors of a treatment with IVM at 0.2 mg kg −1 were cultured in the laboratory and used to infect worm-free sheep in a closed experimental system. http://parasites.trends.com

Such an experimentally selected strain should be distinguished on two grounds from isolates from the field that are ML resistant. First, there were very few survivors of this closed selection process and their genetic diversity would have been greatly restricted, not only in terms of any genes that might be involved with resistance

Review

External environment

Inside worm

TRENDS in Parasitology Vol.17 No.9 September 2001

Cuticle

Ivermectin

Efflux/uptake regulation

AVR-15 GluClα2

Pharynx

unc-7 unc-9

Linking neurone

AVR-14 GluClα3

GLC-1 GluClα1

Extrapharyngeal neurone TRENDS in Parasitology

Fig. 2. Proposed model of mechanism of ivermectin action on the pharynx of Caenorhabditis elegans (modified from Ref. 45). Ivermectin (IVM) in the worm acts on various glutamate-gated chloride channels (GluCls). AVR-15 (GluClα2) is essential for the direct action of IVM on paralysis of the pharynx. AVR-14 (GluClα3) and GLC-1 (GluClα1) are not essential for the action of IVM on the pharynx but contribute to its effects.

but also in the diversity of all genes. This process is known as bottlenecking. Second, if one allele by itself can confer sufficient resistance to allow some of the adult worms to survive, only a single gene will appear to be responsible for resistance. However, there might be several genes that can contribute to ML resistance and, in a field selection process in which several generations of selection might take place before resistance is noticed, breeding between different generations of survivors of treatment and nematodes in refugia will have occurred. More than a single allele on a single gene might have been selected in resistant worms. Where IVM resistance has been found in the field, there have typically been 5–30 treatments with an AVM (Ref. 48). One can also question whether selection over several generations, as typically occurs in the field, but with doses of ML below the recommended dose rate49 also reflects the situation of AVM resistance found in the field. Such selection is likely to select for all of the alleles on all of the genes that can contribute to AVM resistance in the population under selection. Analyses of strains selected in this way are likely to reveal all of the potential resistance-associated genes, but fail to distinguish which gene might have the largest effect in field-selected AVM resistance. Genes implicated in IVM resistance in parasitic nematodes and their possible association with physiological functions are illustrated in Fig. 3. The first gene found to be associated with ML resistance in H. contortus encoded a Pgp, PGP-A (Refs 8,50), which is found in two strains of worms selected with IVM (IVC and IVF17) and one strain selected with MOX (MOF17), compared with their unselected parental strains (BBH in the case of IVC and PF17 in the cases of IVF17 and MOF17). These three selected strains were selected51 (G.T. Wang et al., unpublished) by repeated treatment with sublethal doses of IVM or MOX. Subsequently, it was independently found that http://parasites.trends.com

451

there was selection on the gene for the same Pgp (referred to as A27) and another Pgp (A28) in two experimentally selected IVM-resistant strains (Warren and CAVRS)9. Using the H. contortus–H. placei backcross method, it was found that the gene for another Pgp, hcpgp-1, is selected by IVM in the CAVRS resistant strain35. However, although this gene is associated with IVM resistance in CAVRS, it is not the major gene responsible for resistance in this strain35. It is interesting that the genes encoding Pgps seem to be closely linked with each other in C. elegans52; thus, selection on one or more of these genes during treatment with MLs might select certain alleles of genes for other Pgps because they will segregate with the Pgp-encoding gene(s) that contribute to AVM resistance. Pgp might play a role in removing the MLs from cells containing ML receptors and so it is interesting that IVM is a potent ligand for Pgps (Refs 53,54) and that multidrug resistance reversing agents, which inhibit the transport functions of some Pgps, can partially reverse ML resistance in H. contortus50,55. The overwhelming evidence of an association between Pgps and ML resistance suggests that these genes will be useful markers for ML resistance. Comparison of the polymorphism in ML-selected strains IVC, IVF17 and MOF17 with their parental unselected strains (BBH and PF17) revealed that there was selection on a gene encoding GluCl (Ref. 10), which was subsequently characterized as two alternatively spliced cDNAs, HcGluCla and HcGluClb (Ref. 27). Membranes from IVM-selected strains had higher maximum binding under equilibrium conditions (Bmax) for glutamate binding than did unselected strains56,57, and IVM decreased the Bmax for glutamate binding in a susceptible strain (PF17) but not in the IVF17-selected strain. Furthermore, glutamate attenuates the inhibitory effect on pharyngeal pumping of MOX, but not IVM, in susceptible H. contortus and of both MOX and IVM in IVM-selected H. contortus58. These studies suggest that an upregulation of glutamate binding is involved in IVM resistance, whereas IVM decreased glutamate binding in susceptible H. contortus. In C. elegans and Drosophila melanogaster59,60, IVM potentiates the action of glutamate on channel opening. Nematodes have glutamate reuptake transporters61, which could relieve the effects of glutamate on GluCl channels and also constitute part of the glutamate binding in crude membrane preparations. Part of the action of IVM appears to be the potentiation of glutamate-gated opening of the GluCl channels and a component of the ML-resistance mechanism could be higher levels of glutamate reuptake in the vicinity of the GluCl receptors. Such a model is consistent with the glutamate-binding data, but further work needs to be done to test this hypothesis. HG1, a putative member of the amino acid-gated anion channel subunit family62, has also been found to be selected by IVM (strains IVC and IVF17) and MOX (strain MOF17) compared with their parental

Review

452

TRENDS in Parasitology Vol.17 No.9 September 2001

Conclusions

Pharynx

Uterine muscle

(a)

(c)

Somatic muscle

Linker neurone

(b)

Nerve cord

Interneurone

Key: AVM-sensitive GluCl receptors

Muscle cell

Pgp transmembrane pump GABA receptor

TRENDS in Parasitology

Fig. 3. Proposed model for the action of avermectin (AVM) and milbemycin on parasitic nematodes, involving possible paralysis of the pharynx, somatic and uterine muscles. (a) In the pharynx, pharyngeal muscle cells are connected to the nerve cord by a linking neuron. AVM-sensitive GluCl receptors are on the neuron or the muscle cell. The Pgp transmembrane pump regulates the conentration of AVM in the cell membrane. (b) In the somatic muscle, signals from the nerve cord pass through interneurons to somatic muscle cells. There are possibly AVM-sensitive GluCl receptors and Pgp pumps on neurons and/or muscles cells and AVM-sensitive GABA receptors on interneurons. (c) In the uterine muscle, the uterus or the neurons to the uterus also have receptors sensitive to AVM and possibly Pgp pumps. Abbreviations: GABA, γ-aminobutyric acid; GluCl, glutamate-gated chloride channel; Pgp, P-glycoprotein.

unselected strains (BBH and PF17, respectively) (W.J. Blackhall, PhD thesis, McGill University, Montreal, Canada, 2000). Further work is required to characterize the function of this protein. A number of other genes have been investigated for association with ML selection in H. contortus, including a GluCl β-subunit, an N-acetylcholine-receptor, phosphoenolpyruvate carboxykinase and phosphofructokinase3,10, and none of these genes was linked with ML selection. Another gene of potential interest for ML resistance is the Hc-gbr2, which has high homology to C. elegans avr-14 (gbr-2)28. References 1 Keddie, E.M. et al. (1999) Onchocerca volvulus: limited heterogeneity in the nuclear and mitochondrial genomes. Exp. Parasitol. 93, 198–206 2 Okimoto, R. et al. (1992) The mitochondrial genomes of two nematodes, Caenorhabditis elegans and Ascaris suum. Genetics 130, 471–498 3 Keddie, E.M. et al. (1998) The mitochondrial genome of Onchocerca volvulus: sequence, structure and phylogenetic analysis. Mol. Biochem. Parasitol. 95, 111–127 4 Blouin, M.S. et al. (1995) Host movement and the genetic structure of populations of parasitic nematodes. Genetics 141, 1007–1014 5 Anderson, T.J.C. et al. (1998) Population biology of parasitic nematodes: application of genetic markers. Adv. Parasitol. 41, 219–284 6 Kwa, M.S.G. et al. (1993) Effect of selection for benzimidazole resistance in Haemonchus contortus on β-tubulin isotype 1 and isotype 2 genes. Biochem. Biophys. Res. Commun. 191, 413–419

http://parasites.trends.com

Resistance in H. contortus to LEV, BZ or ML might involve more than one gene for each type of resistance. The genes selected might vary between different isolates, depending on the genetic diversity of the parental population and on the method of selection. LEV resistance is associated with N-acetylcholine-receptors, BZ resistance with β-tubulins, and ML resistance with Pgps, possibly some of the diverse amino acid-gated anion channel subunits found in nematodes and possibly with proteins involved with glutamate transport. Considerable progress has been made recently in obtaining a better understanding of anthelmintic resistance in H. contortus. However, the gaps in our knowledge of the major genes involved in LEV and ML resistance require further research. The existing biological methods of monitoring resistance, such as larval development assays, egg reduction assays and worm reduction assays are too insensitive to monitor the development of resistance before there is a resistance problem. DNA-based assays will provide information on the absolute frequency of resistance alleles even when their frequency is low. Monitoring will allow appropriate advice to be given with full knowledge of the implications of control methods for selection for resistance. In many parts of the world, it is too late to stop the selection for anthelmintic resistant in H. contortus. However, there is still a need to take action to reduce selection for anthelmintic resistance in nematodes of other hosts, such as cattle, in which ML use is increasing, and in human populations in which widespread treatment programs for gastrointestinal nematodes, lymphatic filaria and O. volvulus, using IVM and BZ anthelmintics are commencing. To do this, we need DNA-based tools for anthelmintic resistance in each of the main target parasite species. In addition, molecular studies on anthelmintic resistance will provide insights to new chemotherapeutic and immunotherapeutic means to control parasites.

7 Beech, R.N. et al. (1994) Genetic variability of the β-tubulin genes in benzimidazole-susceptible and -resistant strains of Haemonchus contortus. Genetics 138, 103–110 8 Blackhall, W.J. et al. (1998) Selection at a Pglycoprotein gene in ivermectin- and moxidectinselected strains of Haemonchus contortus. Mol. Biochem. Parasitol. 95, 193–201 9 Sangster, N.C. et al. (1999) Haemonchus contortus: sequence heterogeneity of internucleotide binding domains from P-glycoproteins and an association with avermectin/milbemycin resistance. Exp. Parasitol. 91, 250–257 10 Blackhall, W.J. et al. (1998) Haemonchus contortus: selection at a glutamate-gated chloride channel gene in ivermectin- and moxidectin-selected strains. Exp. Parasitol. 90, 42–48 11 Hoekstra, R. et al. (1999) Characterisation of a polymorphic Tcl-like transposable element of the parasitic nematode Haemonchus contortus. Mol. Biochem. Parasitol. 102, 157–166

12 Greenhalgh, C.J. et al. (1999) Galectins from sheep gastrointestinal nematode parasites are highly conserved. Mol. Biochem. Parasitol. 98, 285–289 13 Hoekstra, R. et al. (2000) Transposon associated markers for the parasitic nematode Haemonchus contortus. Mol. Biochem. Parasitol. 105, 127–135 14 Hoekstra, R. et al. (1997) Microsatellites of the parasitic nematode Haemonchus contortus: polymorphism and linkage with a direct repeat. Mol. Biochem. Parasitol. 89, 97–107 15 Blouin, M.S. et al. (1992) Unusual population genetics of a parasitic nematode; mtDNA variation within and among populations. Evolution 46, 470–476 16 Bamber, B.A. et al. (1999) The Caenorhabditis elegans unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor. J. Neurosci. 19, 5348–5359 17 Lubega, G.W. and Prichard, R.K. (1991) Betatubulin and benzimidazole resistance in the sheep nematode Haemonchus contortus. Mol. Biochem. Parasitol. 47, 129–138

Review

18 Roos, M.H. et al. (1995) New genetic and practical implications of selection for anthelmintic resistance in parasitic nematodes. Parasitol. Today 11, 148–150 19 Geary, T.G. et al. (1993) Haemonchus contortus: ivermectin-induced paralysis of the pharynx. Exp. Parasitol. 77, 88–96 20 Holden-Dye, L. and Walker, R.J. (1990) Avermectin and avermectin derivatives are antagonists at the 4-aminobutyric acid (GABA) receptor on the somatic muscle cells of Ascaris; is this the site of anthelmintic action? Parasitology 101, 265–271 21 Skinner, T.M. et al. (1998) Immunochemical localization of a putative inhibitory amino acid receptor subunit in the parasitic nematodes Haemonchus contortus and Ascaris suum. Parasitology 117, 89–96 22 Kass, I.S. (1984) Effects of avermectin and drugs related to acetylcholine and 4-aminobutyric acid on neurotransmission in Ascaris suum. Mol. Biochem. Parasitol. 13, 213–225 23 Cull-Candy, S.G. and Usherwood, P.N.R. (1973) Two populations of L-glutamate receptors on locust muscle fibres. Nat. New Biol. 246, 62–64 24 Cully, D.F. et al. (1994) Cloning of an avermectinsensitive glutamate-gated chloride channel from Caenorhabditis elegans. Nature 371, 707–711 25 Dent, J.A. et al. (1997) avr-15 encodes a chloride channel subunit that mediates inhibitory glutamergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans. EMBO J. 16, 5867–5879 26 Laughton, D.L. et al. (1997) Reporter gene constructs suggest that the Caenorhabditis elegans avermectin receptor beta-subunit is expressed solely in the pharynx. J. Exp. Biol. 200, 1509–1514 27 Forrester, S.G. et al. (1999) Cloning, sequencing, and development expression levels of a novel glutamate-gated chloride channel homologue in the parasitic nematode Haemonchus contortus. Biochem. Biophys. Res. Commun. 254, 529–534 28 Jagannathan, S. et al. (1999) Ligand-gated chloride channel subunits encoded by the Haemonchus contortus and Ascaris suum orthologues of the Caenorhabditis elegans gbr-2 (avr-14) gene. Mol. Biochem. Parasitol. 103, 129–140 29 Delany, N.S. et al. (1998) Cloning and localisation of an avermectin receptor-related subunit from Haemonchus contortus. Mol. Biochem. Parasitol. 97, 177–187 30 Kotze, A.C. (1998) Effect of macrocyclic lactones on ingestion in susceptible and resistant Haemonchus contortus larvae. J. Parasitol. 84, 631–635 31 Gill, J.H. et al. (1998) Evidence of multiple mechanisms of avermectin resistance in Haemonchus contortus-comparison of selection protocols. Int. J. Parasitol. 28, 783–789

TRENDS in Parasitology Vol.17 No.9 September 2001

32 Grant, W.N. (1994) Genetic variation in parasitic nematodes and its implications. Int. J. Parasitol. 24, 821–830 33 Blaxter, M. and Bird, D. (1997) Parasitic nematodes. In C. elegans II (Riddle, D.L. et al., eds), pp. 851–878, Cold Spring Harbor Laboratory Press 34 Kwa, M.S.G. et al. (1995) Beta-tubulin genes from the parasitic nematode Haemonchus contortus modulate drug resistance in Caenorhabditis elegans. J. Mol. Biol. 246, 500–510 35 Le Jambre, L.F. et al. (1999) A hybridisation technique to identify anthelmintic resistance genes. Int. J. Parasitol. 29, 1979–1985 36 Lewis, J.A. et al. (1980) Levamisole-resistant mutants of the nematode Caenorhabditis appear to lack pharmacological acetylcholine receptors. Neuroscience 5, 967–989 37 Fleming, J.T. et al. (1997) Caenorhabditis elegans levamisole resistance genes lev-1, unc-29 and unc-38 encode functional nicotinic acetylcholine receptor subunits. J. Neurosci. 17, 5843–5857 38 Martin, R.J. et al. (1997) Heterogenous levamisole receptors: a single channel study of nicotinic acetylcholine receptors from Oesophagostomum dentatum. Eur. J. Pharmacol. 322, 249–257 39 Sangster, N.C. et al. (1998) Binding of [3H]maminolevamisole to receptors in levamisolesusceptible and -resistant Haemonchus contortus. Int. J. Parasitol. 28, 707–717 40 Sangster, N.C. et al. (1998) Inheritance of levamisole and benzimidazole resistance in an isolate of Haemonchus contortus. Int. J. Parasitol. 28, 503–510 41 Dobson, R.J. et al. (1996) Management of anthelmintic resistance: inheritance of resistance and selection with persistent drugs. Int. J. Parasitol. 26, 993–1000 42 Lubega, G.W. et al. (1994) Haemonchus contortus: the role of two β-tubulin gene subfamilies in the resistance to benzimidazole anthelmintics. Biochem. Pharmacol. 47, 1705–1715 43 Kwa, M.S.G. et al. (1994) Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation at amino acid 200 in β-tubulin isotype I. Mol. Biochem. Parasitol. 63, 299–303 44 Elard, L. et al. (1999) PCR diagnosis of benzimidazolesusceptibility or -resistance in natural populations of the small ruminant parasite, Teladorsagia circumcincta. Vet. Parasitol. 80, 231–237 45 Dent, J.A. et al. (2000) The genetics of ivermectin resistance in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U. S. A. 97, 2674–2679 46 Gill, J.H. and Lacey, E. (1998) Avermectin resistance in trichostrongylid nematodes. Int. J. Parasitol. 28, 863–877 47 Le Jambre, L.F. et al. (2000) Inheritance of avermectin resistance in Haemonchus contortus. Int. J. Parasitol. 30, 105–111

453

48 Schoop, W.L. (1993) Ivermectin resistance. Parasitol. Today 9, 154–159 49 Egerton, J.R. et al. (1988) Laboratory selection of Haemonchus contortus for resistance to ivermectin. J. Parasitol. 74, 614–617 50 Xu, M. et al. (1998) Ivermectin resistance in nematodes may be caused by alteration of Pglycoprotein homolog. Mol. Biochem. Parasitol. 91, 327–335 51 Rohrer, S.P. et al. (1994) Ivermectin binding sites in sensitive and resistant Haemonchus contortus. J. Parasitol. 80, 493–497 52 Linke, C.R. et al. (1992) The P-glycoprotein gene family of Caenorhabditis elegans. Cloning and characterization of genomic and complementary DNA sequences. J. Mol. Biol. 228, 701–711 53 Didier, A. and Loor, F. (1996) The abamectin derivative ivermectin is a potent P-glycoprotein inhibitor. Anti-cancer Drugs 7, 745–751 54 Pouliot, J-F. et al. (1997) Reversal of Pglycoprotein-associated multidrug resistance by ivermectin. Biochem. Pharmacol. 53, 17–25 55 Molento, M.B. and Prichard, R.K. (1999) Effects of the multidrug-resistance-reversing agents verapamil and CL 347,099 on the efficacy of ivermectin or moxidectin against unselected and drug-selected strains of Haemonchus contortus in jirds (Meriones unguiculatus). Parasitol. Res. 85, 1007–1011 56 Paiement, J.P. et al. (1999) Haemonchus contortus: characterization of a glutamate binding site in unselected and ivermectin-selected larvae and adults. Exp. Parasitol. 92, 32–39 57 Hejmadi, M.V. et al. (2000) L-Glutamate binding sites of parasitic nematodes: an association with ivermectin resistance? Parasitology 120, 535–545 58 Paiement, J-P. et al. (1999) Haemonchus contortus: effects of glutamate, ivermectin, and moxidectin on inulin uptake activity in unselected and ivermectin-selected adults. Exp. Parasitol. 92, 193–198 59 Arena, J.P. et al. (1992) Expression of a glutamate-activated chloride channel current in Xenopus oocytes injected with Caenorhabditis elegans RNA: evidence for modulation by avermectin. Mol. Brain Res. 15, 339–348 60 Cully, D.F. et al. (1996) Identification of a Drosophila melanogaster glutamate-gated chloride channel sensitive to the antiparasitic agent avermectin. J. Biol. Chem. 271, 20187–20191 61 Davis, R.E. (1998) Action of excitatory amino acids on hypodermis and the motor nervous system of Ascaris suum: pharmacological evidence for a glutamate transporter. Parasitology 116, 487–500 62 Laughton, D.L. et al. (1994) Cloning of a putative inhibitory amino acid receptor from the parasitic nematode Haemonchus contortus. Recept. Channels 2, 155–163

Award program in pathogenesis of infectious disease The Burroughs Wellcome Fund (BWF) is offering up to 16 grants to accomplished researchers at the assistant professor level in the USA and Canada. BWF is particularly interested in how human hosts handle infectious disease. Research studies of interest include host–pathogen interactions originating in viral, bacterial, fungal or parasite systems that focus on the effects on the host at the cellular or systemic level. Each award is worth US$400 000 over five years. BWF encourages applications from women and from members of under-represented minority groups. The application deadline for the 2002 awards is 1 November 2001. For more information, please contact Jean Kramarik [tel: + 1 (919) 991 5122], or download the brochures from www.bwfund.org

http://parasites.trends.com