P-glycoprotein-9 and macrocyclic lactone resistance status in selected strains of the ovine gastrointestinal nematode, Teladorsagia circumcincta.

The Teladorsagia circumcincta P-glycoprotein-9 (Tci-pgp-9) gene has previously been implicated in multiple-anthelmintic resistance in this parasite. Here we further characterise genetic diversity in Tci-pgp-9 and its possible role in ivermectin (IVM) and multi-drug resistance using two UK field isolates of T. circumcincta, one susceptible to anthelmintics (MTci2) and the other resistant to most available anthelmintics including IVM (MTci5). A comparison of full-length Tci-pgp-9 cDNA transcripts from the MTci2 and MTci5 isolates (∼3.8 kb in both cases) indicated that they shared 95.6% and 99.5% identity at the nucleotide and amino acid levels, respectively. Nine non-synonymous SNPs were found in the MTci5 sequences relative to their MTci2 counterparts. Twelve genomic sequence variants of the first internucleotide binding domain of Tci-pgp-9 were identified and up to 10 of these were present in some individual worms, strongly supporting previous evidence that amplification of this gene has occurred in T. circumcincta. On average, fewer distinct sequence variants of Tci-pgp-9 were present in individual worms of the MTci5 isolate than in those of the MTci2 isolate. A further reduction in the number of sequence variants was observed in individuals derived from an IVM-treated sub-population of MTci5. These findings suggest that Tci-pgp-9 was under purifying selection in the face of IVM treatment in T. circumcincta, with some sequence variants being selected against.

Genomic introgression mapping of field-derived multiple-anthelmintic resistance in Teladorsagia circumcincta.

Preventive chemotherapy has long been practiced against nematode parasites of livestock, leading to widespread drug resistance, and is increasingly being adopted for eradication of human parasitic nematodes even though it is similarly likely to lead to drug resistance. Given that the genetic architecture of resistance is poorly understood for any nematode, we have analyzed multidrug resistant Teladorsagia circumcincta, a major parasite of sheep, as a model for analysis of resistance selection. We introgressed a field-derived multiresistant genotype into a partially inbred susceptible genetic background (through repeated backcrossing and drug selection) and performed genome-wide scans in the backcross progeny and drug-selected F2 populations to identify the major genes responsible for the multidrug resistance. We identified variation linking candidate resistance genes to each drug class. Putative mechanisms included target site polymorphism, changes in likely regulatory regions and copy number variation in efflux transporters. This work elucidates the genetic architecture of multiple anthelmintic resistance in a parasitic nematode for the first time and establishes a framework for future studies of anthelmintic resistance in nematode parasites of humans.

Real-time PCR/DNA melting curve-based assay to identify individual strongylid larvae recovered from ovine faecal cultures.

A closed-tube real-time PCR (RT PCR) method was developed to identify individual strongylid nematode larvae recovered from ovine faecal cultures. The method builds on an earlier conventional PCR assay established by our group and similarly targets species-specific sequence motifs in the ITS-2 region of ribosomal DNA. The new procedure combines RT PCR with DNA melting curve analyses to identify species-specific amplicons, thus avoiding the need to undertake gel electrophoresis. As with the earlier method, it involves two sets of species-specific reactions. The first targets Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus spathiger and Oesophagostomum venulosum while the second targets Trichostrongylus axei, Trichostrongylus vitrinis, Cooperia curticei and Chabertia ovina. With two exceptions, all the DNA primers employed in the new assay were among those described and tested in developing the earlier assay. The exceptions are the forward "generic" primer, which was re-designed to generate smaller amplicon sizes more suitable for melting curve analyses, and the T. axei-specific primer, which was modified to achieve a higher amplicon melt temperature to enable larvae of this species to be more readily differentiated from those of C. curticei. The melt temperature range for amplicons representing each of the species targeted was determined using lysates derived from both microscopically identified adult male worms (2-12/species), as well as 30 larvae of each of the species which were derived from at least 6 different geographical locations throughout New Zealand. The new assay potentially provides a simpler, faster method to identify individual ovine strongylid larvae for downstream applications than was provided by the earlier conventional PCR assay.

Discovery of quantitative trait loci for resistance to parasitic nematode infection in sheep: I. Analysis of outcross pedigrees.

BACKGROUND: Currently most pastoral farmers rely on anthelmintic drenches to control gastrointestinal parasitic nematodes in sheep. Resistance to anthelmintics is rapidly increasing in nematode populations such that on some farms none of the drench families are now completely effective. It is well established that host resistance to nematode infection is a moderately heritable trait. This study was undertaken to identify regions of the genome, quantitative trait loci (QTL) that contain genes affecting resistance to parasitic nematodes. RESULTS: Rams obtained from crossing nematode parasite resistant and susceptible selection lines were used to derive five large half-sib families comprising between 348 and 101 offspring per sire. Total offspring comprised 940 lambs. Extensive measurements for a range of parasite burden and immune function traits in all offspring allowed each lamb in each pedigree to be ranked for relative resistance to nematode parasites. Initially the 22 most resistant and 22 most susceptible progeny from each pedigree were used in a genome scan that used 203 microsatellite markers spread across all sheep autosomes. This study identified 9 chromosomes with regions showing sufficient linkage to warrant the genotyping of all offspring. After genotyping all offspring with markers covering Chromosomes 1, 3, 4, 5, 8, 12, 13, 22 and 23, the telomeric end of chromosome 8 was identified as having a significant QTL for parasite resistance as measured by the number of Trichostrongylus spp. adults in the abomasum and small intestine at the end of the second parasite challenge. Two further QTL for associated immune function traits of total serum IgE and T. colubiformis specific serum IgG, at the end of the second parasite challenge, were identified on chromosome 23. CONCLUSION: Despite parasite resistance being a moderately heritable trait, this large study was able to identify only a single significant QTL associated with it. The QTL concerned adult parasite burdens at the end of the second parasite challenge when the lambs were approximately 6 months old. Our failure to discover more QTL suggests that most of the genes controlling this trait are of relatively small effect. The large number of suggestive QTL discovered (more than one per family per trait than would be expected by chance) also supports this conclusion.