Determination of Genes Associated with Macrocyclic Lactone Resistance in Parasitic Nematodes

Some methods for assessing genetic variation in parasitic nematodes have been reviewed previously (Grant, 1994). However, a number of recent approaches are particularly relevant to the study of anthelmintic resistance. A common approach to studying associations between different loci and resistance has been to look for DNA sequence differences in independent drug-sensitive and resistant populations of parasitic nematodes by methods such as restriction fragment length polymorphism (RFLP), single-strand conformational polymorphism (SSCP) and direct sequencing. While 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 locus between susceptible and resistant populations that are genetically independent, for example from different geographic locations, may simply be a reflection of the 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 used successfully to compare polymorphism between ivermectin- and moxidectin-selected strains, and parental unselected strains (Blackhall et al., 1998a,b) to elucidate genes associated with ML resistance in H. contortus.

Another approach to identifying genes associated with anthelmintic resistance in H. contortus has been to produce hybrids by crossing anthelmintic-resistant H. contortus with susceptible H. placei (Le Jambre et al., 1999). Hybrid males are sterile, but females can be backcrossed with placei to give a background of placei, but with resistance-associated genes coming from the contortus ancestor following anthelmintic treatment.

Once the backcrosses have been made, the anthelmintic-selected hybrids can be screened for any candidate gene to see if that gene is contortus or placei by sequence analysis and if the contortus sequence occurs at a higher frequency than expected. This can be a definitive means of showing the association of a gene with resistance. Results must, however, be interpreted with some qualifications. The association does not necessarily mean that the gene, showing a frequency significantly different from that expected from random matings, is itself involved in resistance; it may be located near a gene that is involved in resistance and be linked during segregation. Another qualification is that the sequence analysed always be uniquely different between contortus and placei. In the analysis conducted by Le Jambre et al. (1999), approximately 700 bp were sequenced. No polymorphism at all was described for H. placei in this fragment, even though the H. placei population was not constricted by drug selection. This is surprising given the high diversity of unselected H. contortus and other parasitic 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 previously had been subjected to severe bottlenecking during the experimental ivermectin resistance selection protocol. On the basis of the polymorphism observed in the resistant H. contortus strain and the H. placei, two restriction sites were selected for analysis, a ClaI site that was apparently unique to the single placei polymorph identified and another BamHI site that mostly occurred in placei, but was also present in the less frequent contortus allele. Should the H. placei be more diverse in the fragment analysed and contain a low frequency of alleles without the ClaI site, these alleles would be counted as contortus rather than placei, leading to an overestimate of 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 analysed in each population for results to be interpreted unequivocally. A third qualification is that, as with most of the methods discussed for analysing associations between different genes and 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.

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