Anthelmintic Resistance in Haemonchus contortus: History, Mechanisms and Diagnosis.

Haemonchus contortus has shown a great ability to develop resistance to anthelmintic drugs. In many instances, resistance has appeared less than 10years after the introduction of a new drug class. Field populations of this species now show resistance to all major anthelmintic drug classes, including benzimidazoles (BZs), imidazothiazoles and macrocyclic lactones. In addition, resistance to the recently introduced amino-acetonitrile derivative class (monepantel) has already been reported. The existence of field populations showing resistance to all three major drug classes, and the early appearance of resistance to monepantel, threatens the sustainability of sheep and goat production systems worldwide. This chapter reviews the history of the development of resistance to the various anthelmintics in H. contortus and examines the mechanisms utilized by this species to resist the effects of these drugs. Some of these mechanisms are well understood, particularly for BZ drugs, while our knowledge and understanding of others are increasing. Finally, we summarize methods available for the diagnosis of resistance. While such diagnosis currently relies largely on the faecal egg count reduction test, which suffers from issues of expense and sensitivity, we describe past and current efforts to utilize cheaper and less laborious phenotypic assays with free-living life stages, and then describe progress on the development of molecular assays to provide sensitive resistance-detection tests.

Characterization of Haemonchus contortus P-glycoprotein-16 and its interaction with the macrocyclic lactone anthelmintics.

Anthelmintic resistance in veterinary nematodes, including Haemonchus contortus, has become a limitation to maintaining high standards of animal health. Resistance in this parasite, to all drug families including the macrocyclic lactones (MLs) is a serious issue worldwide. Mechanisms of resistance to the MLs appear to be complex and to include the elimination of these compounds by ABC transporter-like proteins present in nematodes. In order to investigate the potential involvement of ABC transporters in ML resistance in H. contortus, we have characterized the functionality of the ABC transporter H. contortus P-glycoprotein-16 (Hco-PGP-16) expressed in mammalian cells. This has included a study of its interaction with different MLs, including the avermectins, abamectin (ABA) and ivermectin (IVM), and the milbemycin, moxidectin (MOX). Hco-PGP-16 transport activity was studied using the fluorophore Rhodamine 123 (Rho 123). Transfected cells expressing Hco-PGP-16 accumulated less than 50% of Rho 123 than control cells, suggesting an active transport of this tracer dye by Hco-PGP-16. The influence of the MLs on the Rho123 transport by Hco-PGP-16 was then investigated. A marked inhibition of Rho123 transport by ABA and IVM was observed. In contrast, MOX showed less effect on inhibition of Rho123 transport by Hco-PGP-16, and the inhibition was not saturable. The difference in the interaction of the avermectins and MOX with Hco-PGP-16 may help explain the slower rate of development of resistance to MOX compared with the avermectins in H. contortus.

Assessment of benzimidazole resistance in Haemonchus contortus in sheep flocks in Ontario, Canada: comparison of detection methods for drug resistance.

In 2011, a field study was conducted to assess drug resistance of gastro-intestinal nematodes in sheep flocks in Ontario, Canada. Benzimidazole resistance in Haemonchus contortus was assessed by genetic analysis of eggs; measurement of resistant allele percentages at codons 167, 198 and 200 in the ß-tubulin gene was determined on pools of H. contortus eggs using pyrosequencing. Susceptibility to benzimidazoles in gastro-intestinal nematodes was also determined using a Faecal Egg Count Reduction Test (FECRT) and a Larval Development Assay (LDA). In total, 16 farms were assessed with the genetic test. Based on resistant allele frequencies, all of the farms (16/16) tested had benzimidazole resistance in H. contortus; the overall percentage of benzimidazole-resistant H. contortus (estimated prior to treatment using the Hardy-Weinberg formula) was 68.5%. The FECRT and LDA were performed on 11 and 13 farms, respectively. Resistance to fenbendazole was detected on 100% (11/11) of the farms where the FECRT was performed. The LDA revealed the presence of thiabendazole resistance in H. contortus in 92% (12/13) of the farms. Estimated percentages of resistant parasites in H. contortus populations obtained with the two biological tests and the genetic test were compared. The results of the genetic test were in agreement with the biological tests and confirmed that benzimidazole resistance in H. contortus is present in Ontario sheep flocks. Differences between the different methods of drug resistance detection are discussed in terms of cost, time and sampling.

Anthelmintic resistance: markers for resistance, or susceptibility?

The Consortium for Anthelmintic Resistance and Susceptibility (CARS) brings together researchers worldwide, with a focus of advancing knowledge of resistance and providing information on detection methods and treatment strategies. Advances in this field suggest mechanisms and features of resistance that are shared among different classes of anthelmintic. Benzimidazole resistance is characterized by specific amino acid substitutions in beta-tubulin. If present, these substitutions increase in frequency upon drug treatment and lead to treatment failure. In the laboratory, sequence substitutions in ion-channels can contribute to macrocyclic lactone resistance, but there is little evidence that they are significant in the field. Changes in gene expression are associated with resistance to several different classes of anthelmintic. Increased P-glycoprotein expression may prevent drug access to its site of action. Decreased expression of ion-channel subunits and the loss of specific receptors may remove the drug target. Tools for the identification and genetic analysis of parasitic nematodes and a new online database will help to coordinate research efforts in this area. Resistance may result from a loss of sensitivity as well as the appearance of resistance. A focus on the presence of anthelmintic susceptibility may be as important as the detection of resistance.

Reduced genetic variation of an Onchocerca volvulus ABC transporter gene following treatment with ivermectin.

In some trichostrongyloid nematodes, the early stages of ivermectin (IVM) resistance have been characterized by a shift in allele frequency and reduced polymorphism at loci of P-glycoprotein genes, glutamate-gated chloride channel genes and gamma-aminobutyric acid receptor genes. Mass treatment with IVM is an integral component of the onchocerciasis control programmes. Genetic variation of an Onchocerca volvulus ABC transporter homologue (OvABC-3) from several populations in Africa was examined to determine whether an association exists between alleles of this gene and IVM treatment. Allelic variation in a non-treated population from Ghana showed this locus to be highly polymorphic. However, variability was reduced in IVM-treated populations. chi2 analysis of polymorph frequencies showed significant differences between untreated and treated samples collected in Ghana in 1999. There was less variability in this gene in samples collected in 2002 compared with the 1999 samples. In some treated populations, there appeared to be selection on OvABC-3-C. The observed reduction in variability could be expected in a control programme in which prevalence and intensity of infections are markedly reduced after years of vector control and IVM distribution. The reduction in polymorphism may not in itself indicate that these O. volvulus are IVM resistant, although it could indicate that selection for resistance is occurring.

Foreword: towards markers for anthelmintic resistance in helminths of importance in animal and human health.

Anthelmintic resistance is a serious problem in veterinary medicine and appears to be developing in some helminths of importance to human health. Anthelmintic drugs remain the principal means of control of helminth infections in animals and humans and the continued dependence on these pharmaceuticals will continue to impose selection pressure for resistance development. Our ability to detect anthelmintic resistance before control breaks down and to monitor the spread of anthelmintic resistance is quite limited. We are currently dependent on biological methods which are not sufficiently sensitive to detect low levels of drug resistance and are particularly difficult to perform on helminth parasites of humans. There is a serious need for new molecular markers for detecting and monitoring for anthelmintic resistance. The problem of anthelmintic resistance is already very serious in nematode parasites of livestock. In addition, there should be great concern about possible anthelmintic resistance development and the lack of tools and efforts for monitoring it as part of the major worldwide programmes to control helminth parasites in people. An international Consortium has been formed to develop Anthelmintic Resistance Single nucleotide polymorphism markers (CARS). Discussions within the Consortium have addressed the need for such markers, the current state of knowledge about them, possible mechanisms of anthelmintic resistance and approaches and constraints to the development of markers. Summaries of the state of the art in these areas are presented in a series of papers in this Special Issue of Parasitology.

ABC transporters and beta-tubulin in macrocyclic lactone resistance: prospects for marker development.

Macrocyclic lactones (MLs) are highly lipophilic anthelmintics which are known to bind to and open ligand-gated ion channels. However, these anthelmintics, and particularly the avermectin members of the ML class of endectocides, are potent substrates for ABC transporters and these transporters may regulate drug concentration in both the host and the parasite. There is accumulating evidence that ivermectin (IVM), and to a lesser extent moxidectin (MOX), selects for certain alleles of P-glycoprotein and other ABC transporter genes, selects for constitutive overexpression of some of these gene products, and induces overexpression of some P-glycoproteins in nematodes. However, such mechanisms of ML resistance do not easily lend themselves to the identification of SNP markers for resistance because of the diversity of ABC transporters in nematodes, the apparent diversity of effects of different MLs, and because regulatory elements for ABC transporter gene expression are not well understood in nematodes. Another non ligand-gated ion channel gene which appears to be under IVM selection, at least in Onchocerca volvulus and Haemonchus contortus, is beta-tubulin, and a simple genetic test for this selection has been described in O. volvulus. However, further work is required to elucidate a reliable marker associated with this gene in H. contortus or other parasitic nematodes of livestock. The possible involvement of ABC transporter genes and beta-tubulin in ML resistance provides a start in developing our understanding of this phenotype and markers for its detection in field populations of parasitic nematodes. However, more work is required before these leads can provide practical SNP markers for ML resistance.

An analysis of the population genetics of potential multi-drug resistance in Wuchereria bancrofti due to combination chemotherapy.

Currently, annual mass treatments with albendazole (ABZ) plus ivermectin (IVM) or diethylcarbamazine (DEC) are administered under the Global Programme to Eliminate Lymphatic Filariasis (GPELF). Drug resistance against both ABZ and IVM is prevalent in nematodes of veterinary importance, raising awareness that if anthelmintic resistance were to develop among Wuchereria bancrofti populations, this would jeopardize GPELF's goals. Genetic structure was incorporated into an existing transmission dynamics model for lymphatic filariasis (LF) to investigate the potential development of concurrent resistance to ABZ and IVM. The resultant models explore the impact of different inheritance modes of resistance to ABZ and IVM on the likely risk of treatment failure under our model assumptions. Results indicate that under ABZ+IVM combination, selection for resistance to one drug is enhanced if resistance to the other drug is already present. Excess parasite homozygosity may increase selection for dominant IVM resistance via enhancing the frequency of recessive ABZ resistance. The model predicts that if multiple resistance genes are associated with different efficacy properties of a drug combination, then examining changes at single loci may be misleading. Sampling schemes in genetic epidemiological surveys investigating the frequency of an allele under selection should consider host age, as individuals of different ages may acquire parasites at different rates.

Ivermectin selection on beta-tubulin: evidence in Onchocerca volvulus and Haemonchus contortus.

Ivermectin resistance is common in trichostrongylid nematodes of livestock, such as Haemonchus contortus. This anthelmintic is the only drug approved for mass administration to control onchocerciasis caused by the nematode parasite, Onchocerca volvulus. In parts of West Africa up to 18 rounds of ivermectin treatment have been administered to communities and there are reports of poor parasitological responses to treatment. Understanding ivermectin resistance and ivermectin selection is an important step to reduce selection pressure for resistance, and to develop molecular markers which can be used to monitor the development of resistance and its spread. Here we report evidence that ivermectin selection changes the frequency of beta-tubulin alleles in both the sheep parasite, H. contortus, and the human parasite, O. volvulus. In O. volvulus we have been able to look at the frequency of beta-tubulin alleles in O. volvulus obtained before any ivermectin was used in humans in Africa, and following its widespread use. In H. contortus, we have been able to look at the frequency of beta-tubulin alleles in a strain which has not seen any anthelmintic selection and in an ivermectin selected strain derived from the unselected strain. We have found ivermectin selects on beta-tubulin in both of these nematode species. In the case of O. volvulus, we had previously reported that ivermectin selects for specific single nucleotide polymorphisms in the O. volvulus beta-tubulin gene. This polymorphism results in three amino acid changes in the H3 helix of beta-tubulin, as well as deletions in an associated intron. We report a simple PCR assay to detect the amplicon length polymorphism, resulting from these intronic deletions, which can be used to monitor the frequency of the beta-tubulin allele selected for by ivermectin in O. volvulus.

Population genetics of concurrent selection with albendazole and ivermectin or diethylcarbamazine on the possible spread of albendazole resistance in Wuchereria bancrofti.

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) intends to achieve its aims through yearly mass treatments with albendazole (ABZ) combined with ivermectin (IVM) or diethylcarbamazine (DEC). The use of ABZ and IVM separately to combat parasites of veterinary importance has, on many occasions, resulted in widespread drug resistance. In order to help predict the spread of potential ABZ resistance alleles through a population of Wuchereria bancrofti, we have developed a mathematical model that incorporates population genetics into EPIFIL, a model which examines the transmission dynamics of the parasite. Our model considers the effect of the combined treatments on the frequency of a recessive allele, which confers ABZ resistance. The model predicts that after 10 yearly treatments with ALB and DEC, 85% coverage and an initial resistance allele frequency of 5%, the frequency of the resistance genotype will increase from 0.25 to 12.7%. If non-random mating is assumed, the initial genotype frequency will be 2.34% and will increase to 62.7%. ABZ and IVM combination treatment may lead to weaker selection for this genotype. Treatment coverage, initial allele frequencies and number of treatments also affect the rate of selection.

The detection of anthelmintic resistance in nematodes of veterinary importance.

Before revised World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines on the detection of anthelmintic resistance can be produced, validation of modified and new methods is required in laboratories in different parts of the world. There is a great need for improved methods of detection of anthelmintic resistance particularly for the detection of macrocyclic lactone resistance and for the detection of resistant nematodes in cattle. Therefore, revised and new methods are provided here for the detection of anthelmintic resistance in nematodes of ruminants, horses and pigs as a basis for discussion and with the purpose that they are evaluated internationally to establish whether they could in the future be recommended by the WAAVP. The interpretation of the faecal egg count reduction test has been modified and suggestions given on its use with persistent anthelmintics and continuous release devices. An egg hatch test for benzimidazole (BZ) resistance is described. A microagar larval development test for the detection of benzimidazole and levamisole resistance provides third stage larvae for the identification of resistant worms. The sensitivity of these two tests can be increased by using discriminating doses rather than LD(50) values. Details are given of a PCR based test for the analysis of benzimidazole resistance in strongyles of sheep and goats, horses and cattle. Although promising for ruminant trichostrongyles, quantitative determination of gene frequency using real time PCR requires further development before PCR tests will be used in the field. Apart from faecal egg count reduction tests there are currently no satisfactory tests for macrocylic lactone resistance despite the great importance of this subject. Except for treatment and slaughter trials there are no validated tests for fasciolicide resistance or for the detection of resistance in cestodes.

Genetic polymorphism of the beta-tubulin gene of Onchocerca volvulus in ivermectin naïve patients from Cameroon, and its relationship with fertility of the worms.

Observations of low response of patients infected with Onchocerca volvulus to ivermectin suggest that the parasite may be under a selection process toward potential resistance. To limit the extension of this phenomenon, it is crucial to characterize the genes of O. volvulus that are involved. For this, O. volvulus adult worms collected before the introduction of ivermectin in an onchocerciasis endemic area of central Cameroon were genotyped for beta-tubulin. To derive a baseline to investigate the selective pressure of ivermectin, we analysed (1) the frequency distribution of the beta-tubulin alleles, and (2) the relationship between the different beta-tubulin related genotypes and the fertility status of the female worms. The frequency of allele b of the beta-tubulin gene was very low, as it was observed in West Africa. We observed a deficit of heterozygous female worms leading to Hardy Weinberg disequilibrium, which might be explained by a shorter life-span of these worms compared to the homozygous worms. Unexpectedly, our results also show that the heterozygous female worms were much less fertile than the homozygotes: more than two thirds of the homozygotes were fertile, whereas only 37% of the heterozygotes were fertile. These results will be further considered when analysing post-treatment data.

Ivermectin imposes selection pressure on P-glycoprotein from Onchocerca volvulus: linkage disequilibrium and genotype diversity.

Widespread use of ivermectin (IVM) as part of the Onchocerciasis Control Program (OCP) in West Africa could influence the evolution of the human filarial parasite Onchocerca volvulus. Use of IVM, in some areas for 15 years, may have restricted genetic diversity of O. volvulus, resembling effects attributed to a population bottleneck. Large population-based chemotherapy programmes, such as the OCP, may impose strong selection pressure on parasites and an examination of possible genetic selection by IVM in O. volvulus is warranted. IVM is a substrate for P-glycoprotein; a homologue from O. volvulus (OvPGP) has been linked with IVM sensitivity. Linkage disequilibrium (LD) patterns of 28 genetic markers spanning the OvPGP locus were examined in 4 O. volvulus populations from the Volta Region of Ghana, West Africa. Reduced gene diversity, increased heterozygosity and an increase in the number of markers not in Hardy-Weinberg equilibrium were associated with increasing IVM treatment. The number of regions in LD decreased with treatment and with time. However, between 1999 and 2002, seven regions of OvPGP were always in complete LD, while surrounding areas showed a reduction in genetic variation. The use of IVM for onchocerciasis control has imposed strong selection on O. volvulus populations, reducing genetic variation and disrupting LD.

Is anthelmintic resistance a concern for heartworm control? What can we learn from the human filariasis control programs?

Heartworm prophylaxis is currently largely dependent on the ability of avermectins and milbemycins to arrest the development of third and fourth stages of Dirofilaria immitis for prolonged periods, without producing adulticidal effects. Major control programs, dependent on the activity of ivermectin, are being implemented for human onchocerciasis and lymphatic filariasis. The avermectins and milbemycins act on glutamate-gated and gamma-aminobutyrate-gated chloride channel subunit proteins in nematodes. Ivermectin resistance has been widely described in trichostrongylid nematodes of ruminants. There is evidence that when ivermectin resistance occurs in nematodes, there may be selection on some, but not all of the genes that code for ligand-gated chloride channel subunit proteins as well as on some ABC-transporter genes, whose products may be involved in regulating macrocyclic lactone drug concentrations at receptors, and on some structural protein genes of amphidial neurones. Although ivermectin resistance has not been reported in filarial nematodes, there have recently been reports of suboptimal responses to ivermectin in Onchocerca volvulus. Evidence has been found of ivermectin selection on at least ABC-transporter genes and some neuronal structural protein genes in O. volvulus. To date, there is no evidence of avermectin/milbemycin resistance in D. immitis, also a filarial nematode. Chemotherapy against trichostrongylids of animals, human filariae, and D. immitis, relies on avermectins or milbemycins. However, control involves targeting different stages or processes in the nematode life cycles, different control strategies, different proportions of the nematode population in refugia, and different drug dosage rates. Consideration of the proportion of the D. immitis population normally in refugia, the life cycle stage targeted, and the anthelmintic dosages used suggest that it is unlikely that significant avermectin/milbemycin resistance will be selected in D. immitis with current treatment strategies.

A comparison of genetic polymorphism in populations of Onchocerca volvulus from untreated- and ivermectin-treated patients.

An analysis of the polymorphism of 16 genes from Onchocerca volvulus was undertaken, in two populations of worms from either ivermectin-naïve patients or patients who had been repeatedly treated with ivermectin, in Ghana. Six genes were selected for analysis because studies in other nematodes had suggested a possible association with ivermectin resistance. The other 10 genes were included as control genes and have not been associated with ivermectin resistance. Twelve of the 16 genes were polymorphic, including five of the candidate genes and seven of the control genes. In all of the control genes and four of the candidate genes, there were no differences in genetic polymorphism between the untreated and ivermectin treatment worms. However, there were statically significant differences (chi2=0.05) in allelic frequencies between the untreated and treatment derived worms for P-glycoprotein and beta-tubulin genes; both genes which have been previously associated with ivermectin resistance in other nematodes. These genes were in Hardy-Weinberg equilibrium in the untreated population. However, the P-glycoprotein alleles, in the worms from the patients under treatment were not in Hardy-Weinberg equilibrium, and analysis of the allele frequencies of beta-tubulin suggested that this gene may have also been under selection in the worms from the ivermectin-treated patients. This data provides evidence of genetic selection by ivermectin on O. volvulus and indicates that investigations should be made to determine whether ivermectin resistance is developing. The beta-tubulin and P-glycoprotein genes may prove useful for monitoring for possible development of ivermectin resistance.

Efficacy of ivermectin in calves against a resistant Cooperia oncophora field isolate.

Since 1999, two Cooperia oncophora isolates, originally obtained from the United Kingdom, have been maintained by regular passage through calves at the Macdonald Campus, McGill University farm. One isolate, IVS, was originally susceptible to ivermectin, while the IVR isolate was originally resistant to ivermectin. These two isolates have been used to study the mechanisms of ivermectin resistance. To confirm the susceptible/resistant status after 4 years of passaging through calves, a controlled study was performed in which two worm-free calves were experimentally infected with IVS and another two worm-free calves with the IVR infective larvae. The calves were treated with ivermectin (0.2 mg/kg) subcutaneously (Ivomec Injection) 21 days after infection. Ivermectin at the recommended dose was 100% effective at eliminating the IVS isolate, since no eggs were found in feces, and no adult worms were found in the small intestine of the treated IVS-infected calf. In contrast, the IVR-infected calf continued to pass eggs in feces even after treatment with ivermectin, and adult worms (250) were found in the small intestine at necropsy. The untreated calves had 1,330 and 848 adult worms, respectively, for the IVS and IVR infected animals.

Identification of variant ABC-transporter genes among Onchocerca volvulus collected from ivermectin-treated and untreated patients in Ghana, West Africa.

Treatment with ivermectin (IVM) is known to cause a loss of polymorphism at certain loci of the beta-tubulin, gamma-aminobutyric-acid-receptor, glutamate-gated-chloride-channel and ATP-binding-cassette (ABC) transporter genes of IVM-resistant Haemonchus contortus. The genetic variation of four ABC-transporter homologues from Onchocerca volvulus was therefore investigated, to determine if any change in genetic polymorphism occurs in these genes following repeated treatment with IVM. Samples were collected in the Northern, Brong-Ahafo and Volta regions of Ghana, in 1999 and 2002; nodules containing adult O. volvulus were removed from subjects who had either received multiple IVM treatments or never taken IVM. The ATP-binding domains of four ABC-transporter genes (OvMDR-1, OvMDR-3, OvABC-1 and OvABC-6) were amplified from individual O. volvulus and examined for polymorphism, using single-strand-conformation-polymorphism (SSCP) analysis. In the samples collected in 1999, OvMDR-1 and OvABC-1 showed significant reduction in polymorphism following IVM treatment whereas OvABC-6 and OvMDR-3 were not found to be polymorphic. The samples collected in 2002 also showed a reduction in polymorphism for both OvMDR-1 and OvABC-1. Several single-nucleotide polymorphisms, which resulted in either amino-acid-replacement substitutions or nonsense mutations, were identified in the alleles of OvMDR-1 and OvABC-1.

Genetic variability in cysteine protease genes of Haemonchus contortus.

To increase the existent genetic variability in cysteine proteases, a polymorphism study was performed in Haemonchus contortus by comparing 2 different strains of the parasite: North American (NA) and Spanish (SP) strains. For this purpose, the polymorphism of 5 previously reported genes (AC-1, AC-3, AC-4, AC-5 and GCP-7) were analysed by PCR-SSCP and sequencing procedures. Based on the SSCP results, a total of 20 different alleles were identified for the 5 loci assessed. Except locus AC-5, all the loci were polymorphic. Loci AC-1, AC-3, AC-4 and GCP-7 showed 5, 8, 2 and 4 alleles, respectively. The allelic frequencies ranged from 0.0070 to 0.8560 and were significantly different between strains. In addition, nucleotide diversity analyses showed a significant variation within and between strains. The variations in the nucleotide sequence of the different alleles were translated in some cases into changes in the amino acid sequence. Evidence of genetic variability in cysteine proteases from two different strains of H. contortus for the same set of genes had not been previously reported.

Genetic variability of glutamate-gated chloride channel genes in ivermectin-susceptible and -resistant strains of Cooperia oncophora.

The glutamate-gated chloride channels (GluCls) are members of the ligand-gated ion channel superfamily that are thought to be involved in the mode of action of ivermectin and mechanism of resistance. Using reverse-transcriptase PCR techniques, 2 full-length GluCl cDNAs, encoding GluClalpha3 and GluClbeta subunits, were cloned from Cooperia oncophora, a nematode parasite of cattle. The two sequences show a high degree of identity to similar subunits from other nematodes. The C. oncophora GluClalpha3 subunit is most closely related to the Haemonchus contortus GluClalpha3B subunit, while C. oncophora GluClbeta subunit shares high sequence identity with the H. contortus GluClbeta subunit. Using single-strand conformation polymorphism, the genetic variability of these two genes was analysed in an ivermectin-susceptible isolate and an ivermectin-resistant field isolate of C. oncophora. Statistical analysis suggested an association between the C. oncophora GluClalpha3 gene and ivermectin resistance. No such association was seen with the GluClbeta gene.

Cloning two full-length beta-tubulin isotype cDNAs from Cooperia oncophora, and screening for benzimidazole resistance-associated mutations in two isolates.

Two full-length beta-tubulin cDNAs, representing isotypes 1 and 2, were cloned from the cattle nematode Cooperia oncophora. The predicted protein sequences span 448 amino acids, and show a high degree of identity to beta-tubulins from other nematodes. While C. oncophora isotype 1 sequence had the highest identity to Haemonchus contortus isotype 1 and Teladorsagia circumcincta sequences (95% identity), the C. oncophora isotype 2 sequence was most similar to H. contortus isotype 2 and Trichostrongylus colubriformis (92% identity). Alignment of the two C. oncophora sequences with other trichostrongylid beta-tubulins deposited in GenBank showed a clear distinction between isotype 1 and 2 beta-tubulin classes. The two classes differed at 19 amino acid positions, most notably at the carboxy terminus. These isotype-defining residues were conserved among different trichostrongylid species within a class. Analysis of fragments of both genes revealed a high degree of genetic variability in coding and non-coding regions. However, all nucleotide differences detected in the coding region were silent, as they did not result in any amino acid substitution. Analysis of 2 groups of worms for the codon 200 polymorphism associated with benzimidazole resistance revealed a proportion of worms in 1 of the groups bearing a tyrosine at this position.