SNP MARKERS ASSOCIATED WITH BZ RESISTANCE IN TRICHOSTRONGYLIDS

Work in the early 1990s identified genetic changes in genes encoding β-tubulins as the likely cause of BZ resistance in H. contortus (Roos et al. Reference Roos, Boersema, Borgsteede, Cornelissen, Taylor and Ruitenberg1990). Two genes, isotypes 1 and 2, were isolated (Geary et al. Reference Geary, Nulf, Favreau, Tang, Prichard, Hatzenbuhler, Shea, Alexander and Klein1992) and correlated with resistance (Kwa et al. Reference Kwa, Kooyman, Boersema and Roos1993; Kwa, Veenstra and Roos, Reference Kwa, Veenstra and Roos1993). Further work identified the presence of a SNP, a thymine-to-adenine transversion, in the isotype 1 gene that correlated specifically with BZ resistance (Kwa, Veenstra and Roos, Reference Kwa, Veenstra and Roos1994). This transversion caused the substitution of phenylalanine with tyrosine at codon 200 of the isotype 1 protein. A similar substitution at this position confers resistance to benomyl in a variety of fungi (Kwa et al. Reference Kwa, Veenstra, Van Dijk and Roos1995). Transfection of a BZ-resistant H. contortus allele containing the F200Y polymorphism into the free-living nematode Caenorhabditis elegans conferred BZ resistance on the transfected worms (Kwa et al. Reference Kwa, Veenstra, Van Dijk and Roos1995), verifying this polymorphism as a mechanism of BZ resistance. The F200Y mutation is also the dominant BZ-resistance marker in some other trichostrongylid species, including Teladorsagia circumcincta and Trichostrongylus colubriformis (Grant and Mascord, Reference Grant and Mascord1996; Silvestre and Humbert, Reference Silvestre and Humbert2002).

Initially, the F200Y SNP appeared to be the only polymorphism that led to BZ resistance. However, there is increasing evidence that the situation is not so clear cut, and other loci and/or mechanisms may be involved (Prichard, Reference Prichard2001; Samson-Himmelstjerna et al. Reference Samson-Himmelstjerna, Buschbaum, Wirtherle, Pape and Schnieder2003). For example, highly resistant populations of H. contortus were shown to have also lost the β-tubulin isotype-2 gene (Kwa et al. Reference Kwa, Veenstra and Roos1993; Kwa, Veenstra and Roos, Reference Kwa, Kooyman, Boersema and Roos1993), although this phenomenon has not been observed in all species. Another polymorphic site, position 167, first characterized in fungi (Li, Katiyar and Edlind, Reference Li, Katiyar and Edlind1996; Orbach, Porro and Yanofsky, Reference Orbach, Porro and Yanofsky1986; Thomas, Neff and Botstein, Reference Thomas, Neff and Botstein1985), was subsequently found to be associated with BZ resistance in H. contortus (Prichard et al. Reference Prichard, Oxberry, Bounhas, Sharma, Lubega and Geary2000; Prichard, Reference Prichard2001). As with codon 200, this polymorphism involves a substitution of phenylalanine with tyrosine, but also a substitution of phenylalanine with histidine. In two French populations of H. contortus, F167Y was found only in worms that possessed the wild-type F200 (Silvestre and Cabaret, Reference Silvestre and Cabaret2002). The polymorphism at codon 200 is likely to be the principal cause of BZ resistance in H. contortus field populations (Silvestre and Cabaret, Reference Silvestre and Cabaret2002), but the F167Y polymorphism can attain substantial frequencies in some populations (see below). The F167Y mutation has subsequently been identified in T. circumcincta, but appears to behave differently to that in H. contortus (Silvestre and Cabaret, Reference Silvestre and Cabaret2002). T. circumcincta parasites that are homozygous for the susceptible allele at position 200 express BZ resistance if they are either homo- or heterozygous for the resistant allele at position 167 (Silvestre and Humbert, Reference Silvestre and Humbert2002).

Finally, recent evidence suggests that a third mutation in isotype 1 can also contribute to BZ resistance in H. contortus. Ghisi, Kaminsky and Maser (Reference Ghisi, Kaminsky and Maser2007) have identified an adenine-to-cytosine transversion that leads to a glutamate-to-alanine polymorphism at codon 198 in BZ-resistant populations from Australia and South Africa. Mutations in this codon have been associated with benomyl resistance in a variety of species of fungi (see Ghisi et al. Reference Ghisi, Kaminsky and Maser2007 for a brief discussion). As with F167Y, the E198A polymorphism was found only in worms that were genotypically wild-type at position 200. Despite small sample sizes, the results of this study suggest that E198A occurs at substantial frequencies in the two populations in which it was found. In support of the above study, we have found preliminary evidence for the existence of E198A, resulting from a similar adenine-to-cytosine transversion, in a BZ-resistant population of H. contortus from Germany. The frequency of this polymorphism in the resistant population is estimated to be only about 5% (see below).

Anthelmintic resistance has been slower to develop in cattle parasites than in sheep, even though very similar species infect both hosts. Nonetheless, anthelmintic resistance in cattle is on the increase (Jackson et al. Reference Jackson, Townsend, Pyke and Lance1995; McKenna, Reference McKenna1996) and now represents a serious challenge to the cattle industry. While there have been relatively few investigations into the molecular genetics of resistant cattle parasites, the same genetic determinants appear to be involved, at least in BZ resistance. Using conserved β-tubulin PCR primers, Winterrowd et al. (Reference Winterrowd, Pomroy, Sangster, Johnson and Geary2003) amplified partial genomic sequences from Cooperia oncophora and Ostertagia ostertagi and demonstrated that BZ resistance in field isolates of C. oncophora was associated with the F200Y mutation. Njue and Prichard (Reference Njue and Prichard2003) subsequently cloned and sequenced full-length cDNAs representing both isotypes of β-tubulin from C. oncophora and found a small proportion of individuals that carried the resistant isotype 1 allele (∼24% heterozygous, 3% homozygous). There was no evidence of F167Y or A198E in these studies.

SNP MARKERS ASSOCIATED WITH BZ RESISTANCE IN CYATHOSTOMINS

Several recent field studies have demonstrated that the extent of BZ resistance in cyathostomins has reached drastic levels, with at least 50%, and in some countries >90%, of tested farms showing signs of resistance (Cirak, Gulegen and Bauer, Reference Cirak, Gulegen and Bauer2004; Kaplan et al. Reference Kaplan, Klei, Lyons, Lester, Courtney, French, Tolliver, Vidyashankar and Zhao2004; Meier and Hertzberg, Reference Meier and Hertzberg2005; Varady, Konigova and Corba, Reference Varady, Konigova and Corba2000; Wirtherle, Schneider and Samson-Himmelstjerna, Reference Wirtherle, Schnieder and Samson-Himmelstjerna2004). However, research into the prevalence of BZ resistance is hampered by the lack of inexpensive, sensitive and reliable diagnostic tools. The commonly used faecal egg count reduction test is relatively time-consuming and laborious. Furthermore, this test is capable of detecting resistance only after >50% of the nematode population carries resistance alleles (Martin, Anderson and Jarrett, Reference Martin, Anderson and Jarrett1989). Improved tests need to be much more sensitive to permit efficient assessment of the resistance level in a population. The analysis of resistance-related allele frequencies in DNA isolated from representative pools of parasites represents such an opportunity (Samson-Himmelstjerna and Blackhall, Reference Samson-Himmelstjerna and Blackhall2005). However, this is only possible if resistance-related genomic markers are known and if their particular significance for the resistance phenotype is understood. A first step in obtaining such markers is the identification and characterization of the nucleotide sequence for the respective target genes in the relevant species. Achieving this goal is likely to be difficult for cyathostomins, which include >50 species having only minor morphological differences and no single species isolate available for experimental work. Accordingly, β-tubulin sequence data have been published for <10 cyathostomin species. The first reported cyathostomin tubulin sequence was the β-tubulin isotype 1 sequence of Cylicocyclus nassatus (Pape, Samson-Himmelstjerna and Schneider, Reference Pape, Samson-Himmelstjerna and Schnieder1999). This was obtained following sequencing of cDNA prepared from pools of adults from a field population for which the treatment history was unavailable. Similar to the previously described β-tubulin sequences of various trichostrongylid species, the C. nassatus sequence encoded 448 amino acids. When aligned with β-tubulin isotype 1 sequences of H. contortus, T. circumcincta and T. colubriformis, identities of >95% were found. Sequencing genomic DNA from individual worms identified both the TTC and TAC codon 200 alleles. The same SNP was detected in the orthologous C. coronatum sequence (Samson-Himmelstjerna et al. Reference Samson-Himmelstjerna, Harder, Pape and Schnieder2001). These were the first indications that the β-tubulin isotype 1 codon 200 TAC allele, which was shown to play a significant role in the mechanism of BZ resistance in H. contortus, T. circumcincta and T. colubriformis (for review see Prichard, Reference Prichard2001), is also present in cyathostomins. Based on the β-tubulin isotype 1 sequence data obtained for C. nassatus, it was possible to design an allele-specific PCR for the analysis of the codon 200 polymorphism (Samson-Himmelstjerna et al. 2002 a). This procedure was suitable for genotyping individual adults and larvae from seven different cyathostomin species. The β-tubulin isotype 1 sequence diversity of these species was further characterized by sequencing at least two full length cDNA clones generated by amplification of mRNA from individual worms isolated from an anthelmintic-naïve cyathostomin population (Pape, Schneider and Samson-Himmelstjerna, Reference Pape, Schnieder and Samson-Himmelstjerna2002). Among the 16 full-length clones obtained, 10 non-synonymous sequence polymorphisms were present, of which only the codon 200 SNP had been previously correlated with BZ resistance. In the same study, attempts to identify a second β-tubulin isotype failed. However, more recently this was achieved for C. nassatus and Cyathostomum catinatum by Clark et al. (Reference Clark, Kaplan, Matthews and Hodgkinson2005), who described intraspecific isotype 2 identities of 94 and 93%, respectively. These data were generated using mRNA isolated from pools of 10 worms from a cyathostomin population with unknown BZ-resistance status. The only known BZ-resistance associated SNP found in this study was at codon 167 in the C. catinatum β-tubulin isotype 1 sequence.

The frequency of the BZ-resistance related codon 200 TAC allele was determined for a BZ-susceptible and an experimentally selected BZ-resistant cyathostomin population (Pape et al. Reference Pape, Posedi, Failing, Schnieder and Samson-Himmelstjerna2003). Within the phenotypically BZ-resistant cyathostomin population, >50% of the 104 adult worms analysed, belonging to six different species, showed the BZ-susceptible TTC homozygous genotype. This finding contrasts with the situation in several small ruminant gastro-intestinal nematodes, in which BZ-resistant populations usually have >90% TAC homozygous worms (see preceding section). Nevertheless, the TAC allele frequency increased significantly during the BZ-resistance selection process achieved by repeated sub-therapeutic and therapeutic BZ treatment in the above mentioned cyathostomin population. Subsequent studies examined the effect of long-term repeated BZ treatment, using therapeutic and up to 4× overdosage fenbendazole (FBZ) treatments, on the BZ-resistance pheno- and genotype of the previously generated BZ-selected cyathostomin population (Drogemuller et al. Reference Drogemuller, Failing, Schnieder and Samson-Himmelstjerna2004). This population had a strong BZ-resistant phenotype, with no faecal egg count reduction following 4× FBZ dosage and an LD₅₀ value >0·36 μg/ml for thiabendazole in the egg hatch test. However, the β-tubulin isotype 1 codon 200 TAC allele frequency remained <40%. This strongly suggests that additional polymorphisms or other mechanisms conferring BZ resistance are present in the cyathostomins. Further attempts were made to identify other BZ-resistance related SNPs in cyathostomins by determining the complete β-tubulin isotype 1 coding sequences of six cyathostomin species isolated from the long-term selected population (Drogemuller, Schneider and Samson-Himmelstjerna, Reference Drogemuller, Schnieder and Samson-Himmelstjerna2004). In up to two species, one to three of the following four β-tubulin isotype 1 codons, found to be correlated with BZ resistance in other organisms, were polymorphic: 6, 165, 167 and 198. However, only the SNP at codon 167, leading to the change from phenylalanine to tyrosine, was detected in all six species studied. The allele frequency and thus significance of this SNP in BZ-susceptible and -resistant cyathostomin species still needs further analysis. Furthermore, it would be informative to investigate the consequences of the alteration in β-tubulin positions 167, 200 (and possibly others) on the BZ-binding affinities of cyathostomin species, as has been done partially for trichostrongylid species (see preceding section). The expression of a recombinant C. nassatus wild-type allele and alleles with the 167 and 200 phenylalanine-tyrosine mutations has recently been achieved (Blackhall et al. Reference Blackhall, Drogemuller, Schnieder and Samson-Himmelstjerna2006). However, the analysis of the β-tubulin/BZ binding has yet to yield informative results, possibly due to misfolding of the protein after solubilisation from inclusion bodies.

SNP MARKERS ASSOCIATED WITH BZ RESISTANCE IN HOOKWORMS

While hookworms of the nematode family Ancylostomatidae are important intestinal parasites of a wide range of mammals, most attention has focused on the species that infect companion animals (dogs and cats) and humans. Clinical manifestations range from growth retardation to life threatening anaemia and protein loss. For this reason, blanket routine control with anthelmintics is widely practised in dogs and cats (http://www.cdc.gov/NCIDOD/DPD/parasites/ascaris/prevention.htm), and is being increasingly advocated in humans (Hotez et al. Reference Hotez, Molyneux, Fenwick, Ottesen, Ehrlich Sachs and Sachs2006). Such chemotherapeutic strategies are believed to promote the evolution of drug resistance. To date, reports of anthelmintic resistance in hookworms are rare. However, pyrantel resistance has been recently reported in the canine parasite Ancylostoma caninum (Kopp et al. Reference Kopp, Kotze, McCarthy and Coleman2007) as well as in human hookworms (Reynoldson et al. Reference Reynoldson, Behnke, Pallant, Macnish, Gilbert, Giles, Spargo and Thompson1997). The molecular basis of pyrantel resistance remains to be elucidated, but since the drug acts as a nicotinic cholinergic ligand, the mechanism of resistance is highly likely to be unrelated to that causing BZ resistance. To date, no molecular analysis of the mechanisms of resistance to nicotinic agonists in hookworms has been published. However, some work has been undertaken to explore the mechanism of resistance in C. elegans and in the porcine parasite Oesophagostomum dentatum (Martin et al. Reference Martin, Verma, Levandoski, Clark, Qian, Stewart and Robertson2005).

Clinical evidence of BZ resistance has yet to be reported in veterinary hookworms. However, BZ resistance in human hookworms has been reported in two studies, one undertaken in Mali (De Clercq et al. Reference De Clercq, Sacko, Behnke, Gilbert, Dorny and Vercruysse1997), and the other on Pemba island, Zanzibar (Albonico et al. Reference Albonico, Bickle, Ramsan, Montresor, Savioli and Taylor2003). These studies have been criticized for methodological limitations (Geerts and Gryseels, Reference Geerts and Gryseels2000), particularly their reliance on historical data for comparing earlier (higher) cure rates to subsequently diminished cure rates. However, studies in human populations are inherently more difficult than those possible in livestock, for which experimental parameters can be tightly controlled and terminal euthanasia to accurately determine efficacy is possible. Further, large-scale chemotherapy programmes with the BZ anthelmintics mebendazole and albendazole are in a relatively early stage compared to the long experience with them in animal husbandry. Only with the formation of the Global Alliance to Eliminate Lymphatic Filariasis (GAELF) (Ottesen, Reference Ottesen2006), and advocacy for the implementation of a so-called ‘Rapid Impact Package’ (Hotez et al. Reference Hotez, Molyneux, Fenwick, Ottesen, Ehrlich Sachs and Sachs2006) for control of neglected tropical diseases are we seeing the prospects of mass distribution of BZ anthelmintics on a scale that approaches that which occurred in livestock, and which ultimately led to the generation of resistance.

For many reasons, including those alluded to above, as well as a relative lack of funding support, systematic analysis of the β-tubulin gene of hookworms has lagged behind that undertaken for intestinal nematodes of importance in livestock industries. In the only published study of β-tubulin sequences in hookworms, Albonico, Wright and Bickle (Reference Albonico, Wright and Bickle2004) analyzed Necator americanus isolates from Pemba island Zanzibar, where clinical resistance had been reported. The authors reported no evidence of the F200Y SNP among 71 individual larvae obtained from a population of 116 children who had received up to 15 rounds of mebendazole therapy. The presence of other possible SNPs in the β-tubulin gene, including the 167 and 198 loci, was not reported. With the growing interest in mass drug administration of BZ anthelmintics to humans, there is a need to undertake systematic study of the β-tubulin genes of the major nematode parasites affecting humans. The significance of this issue is highlighted by the recent report of selection of the F200Y SNP in the lymphatic filarial parasite following albendazole therapy (Schwab et al. Reference Schwab, Boakye, Kyelem and Prichard2005). Likewise, the widespread use of BZs in companion animals, particularly when administered on a large scale and in regular dosing, is likely to select for BZ resistance, particularly since BZ-resistant alleles have been identified in most nematode species studied.

STRATEGIES FOR THE IDENTIFICATION AND ANALYSIS OF RESISTANCE-RELATED SNPs

One of the proposed goals of CARS is to develop genotyping assays for SNPs or other markers that associate with anthelmintic resistance. Various PCR-based assays currently exist for detecting the presence of the polymorphisms in genes encoding β-tubulin isotype 1 that have been associated with BZ resistance (Table 1). A robust allele-specific PCR assay has been developed to genotype individual trichostrongylid nematodes with respect to their BZ resistance status (Elard and Humbert, Reference Elard and Humbert1999; Humbert et al. Reference Humbert, Cabaret, Elard, Leignel and Silvestre2001; Silvestre and Humbert, Reference Silvestre and Humbert2000). These studies have proven extremely informative as to the importance and likely origin of the BZ-conferring mutation in field isolates. For example, a detailed survey of the diversity of BZ-resistance alleles in closed dairy goat farms in Central/Southern France revealed that the F200Y mutation was carried on a number of different alleles in H. contortus, T. circumcincta and T. colubriformis (six, eight and one, respectively). We have employed the same allele-specific PCR to monitor changes in allele frequencies within a triple resistant T. circumcincta isolate following treatment with BZ. Initial findings revealed that a small percentage of survivors of treatment had the susceptible (TTC) genotype, and this was also observed in vitro in eggs capable of hatching in a discriminating dose of BZ (Stenhouse, L., unpublished). Again, this finding indicates that other mutations and/or mechanisms are involved in the expression of BZ resistance. Despite their obvious utility, these techniques rely on agarose gel electrophoresis of multiple individuals to produce meaningful allele frequency readouts. This methodology is both time consuming and labour intensive. To overcome this drawback, a real-time PCR assay has been developed to identify homo- and heterozygous individuals; it can also provide an estimate of allele frequency from pooled samples (Samson-Himmelstjerna et al. Reference Samson-Himmelstjerna, Buschbaum, Wirtherle, Pape and Schnieder2003; Walsh et al. Reference Walsh, Donnan, Jackson, Skuce and Wolstenholme2007). Even this method may already be superseded by the application of pyrosequencing technology (Samson-Himmelstjerna and Blackhall, Reference Samson-Himmelstjerna and Blackhall2005). This is a new method for rapid, high throughput sequence analysis and SNP detection (Troell et al. Reference Troell, Mattsson, Alderborn and Hoglund2003) and is designed to provide accurate allele frequency determinations from multiple individuals but specifically also from pooled samples.

Table 1.

Publications reporting methods for genotyping SNPs associated with BZ resistance

The sequencing of PCR products generated from pooled DNA is a technique commonly used to estimate the frequencies of SNPs in populations. An advantage of this approach is that it can yield an accurate estimate of the relative frequency of each allele in nematode populations. However, this population-based approach may not provide complete information on the relative role of multiple polymorphisms in conferring resistance. For example, if alternative alleles are present in a population at codons 167, 198 and 200, a population-based approach cannot indicate which of the eight possible haplotypes, or combinations thereof, is responsible for the resistant phenotype. We are adapting this technique to rapidly identify SNP genotypes in nematode populations. Here we present preliminary results on SNPs that are associated with BZ resistance.

Genomic DNA was extracted from a pool of 1000 third-stage larvae from each of two field populations of H. contortus from northern Germany. One population was fully sensitive to BZ treatment, whereas the second population was partially resistant. PCR primers were designed to amplify the region of the gene encoding β-tubulin isotype 1 containing the polymorphisms associated with BZ resistance, described above. The two PCR products, one from each population, were then sequenced. Details of the methodology are available from the authors upon request.

Figs. 1 and 2 show the results. The appearance of a secondary peak at a nucleotide site in combination with a reduction in the height of the primary peak at that site, as compared to the peak heights at the same position in the BZ-sensitive population, identifies a SNP in the resistant population. SNPs are indicated by the arrows in the figures. SNP frequencies in the resistant population can be estimated by relative peak heights. Two peaks of equal height indicate the presence of the two polymorphs at equal frequencies in the population, i.e. 0·5 each, as is the case for codon 200 in Fig. 2. If one peak is half the height of the other, as in codon 167 in Fig. 1, the frequency of the lower polymorph is about 0·33. In general, the frequency of a polymorph is estimated by dividing its peak height by the sum of the two peak heights. Applying this method, the frequency of the cytosine polymorph in codon 198 in the population is estimated to be 5%. The estimates of SNP frequencies for codons 167 and 200 shown here agree well with estimates derived from genotyping the same populations, either as multiple individual parasites or as pooled samples, using pyrosequencing (Skuce et al. unpublished). The presence or absence of the polymorphism at codon 198 has not yet been examined by an alternative assay. No evidence of the equivalent mutations at codons 167 and 198 were observed in a similar analysis of BZ-resistant isolates of T. circumcincta (Skuce et al. unpublished).

Fig. 1.

Chromatograms of PCR products in the region of codon 167, indicated by the horizontal bars. The upper trace is from the BZ-sensitive population. The lower trace is from the BZ-resistant population. The thymine-to-adenine transversion in the resistant population is indicated by the arrow.

Fig. 2.

Chromatograms of PCR products in the region of codons 198 and 200, indicated by the horizontal bars. The upper trace is from the BZ-sensitive population. The lower trace is from the BZ-resistant population. The adenine-to-cytosine transversion in codon 198 and the thymine-to-adenine transversion in codon 200 in the resistant population are indicated by the arrows.

The technique described above is a rapid method for genotyping known SNPs in populations. It may also have utility in the discovery of unknown SNPs. Subjecting the respective DNA chromatograms to comparative SeqDoc analysis (Crowe, Reference Crowe2005) effectively subtracts the noise between two pooled samples and facilitates the identification of potentially new mutations associated with the resistant phenotype (Fig. 3). One note of caution, however: populations used for association studies, regardless of how they are analysed, must be matched genetically as closely as possible. False positive signals can arise from a number of sources, most of which are due to genetic differences between the populations unrelated to the phenotype under investigation (for a general discussion of important issues see Newton-Cheh and Hirschhorn, Reference Newton-Cheh and Hirschhorn2005).

Fig. 3.

Comparative SeqDoC analysis of β-tubulin isotype 1 PCR amplified from RNA extracted from ∼1000L1 larvae of T. circumcincta from a BZ-susceptible and BZ-resistant isolate. The P200 (TTC-TAC) mutation is clearly identified.

The rapid advances in SNP discovery and quantification that have occurred as a result of the need to use SNP data in population genetics offer the possibility of applying high throughput approaches to quantitative allelotyping without the need for laborious sequence analysis of individual nematodes. We recently adapted one such technique, mass spectroscopy of allele-specific primer extension products, using the Sequenom® platform. Using this approach, we genotyped two populations of H. contortus with different BZ susceptibility profiles at codons 167, 198 and 200, yielding results that are in close agreement with those obtained by sequencing individual nematodes (McCarthy et al. unpublished). Such approaches offer the possibility of obtaining population level data without the need for the more time consuming approaches. Given the rapid evolution of such technology, it remains to be seen which of the several approaches available for SNP discovery and quantification will prove the most suitable for investigating the genetic basis of drug resistance in helminths.

The genotyping assays referred to in Table 1 are suitable for research purposes and small-scale diagnostics. Pyrosequencing may offer an increase in throughput. Many platforms for genotyping SNPs have been developed in the last decade that promise reliability, reduced cost, and increased throughput. Adapting these new technologies for detecting SNPs associated with BZ, or other anthelmintic, resistance could be useful for the scientific establishment in the future.

In summary, three SNPs are known to be associated with resistance to BZ anthelmintics. The occurrence and degree of contribution to resistance of different SNPs appears to vary among the different species of nematodes and between different populations of the same species. Multiple resistance-associated SNPs occur within single populations of a species but have not yet been found within single worms. The presence of these SNPs has allowed the development of PCR-based assays for genotyping populations or individual worms. Further insight is needed on the prevalence of the individual SNPs and their contribution to the resistance phenotype. This must be investigated in a number of populations of each of the major parasitic nematode species, since intra- and interspecific differences are known to exist. Future studies will also continue the search for other potential causative SNPs and non-causative but resistance-associated SNPs that could be used for a next-generation genotyping assay.