Background Haemonchus contortus , a gastrointestinal nematode parasite of sheep, is mainly controlled by anthelmintics; the occurrence of anthelmintic resistance leads to treatment failures and increases economic burden. Because molecular mechanisms involved in drug resistance can be elucidated by genomic studies, an extreme quantitative trait locus (X-QTL) mapping approach was used to identify co-segregation of the resistance phenotype with genetic markers to detect the genome-wide variants associated with monepantel resistance in H. contortus . Methods A cross between H. contortus isolates using parental susceptible (Par-S) males and monepantel resistant (Par-R) females resulted in SR progeny, while reciprocal cross resulted in RS progeny. Pools ( n = 30,000) of infective larvae (L3) recovered from Par-R, and from SR and RS populations in the F3 generation, collected both before (unselected group) and 7 days after (selected group) selection with monepantel treatment in sheep hosts, were subjected to genome sequencing (Pool-Seq). Pairwise comparisons of allele frequencies between unselected and selected groups were performed for each population by Fisher’s exact test (FET) and for both populations combined by a Cochran-Mantel-Haenszel (CMH) test. Results Mapping rates varied from 80.29 to 81.77% at a 90.4X mean coverage of aligned reads. After correction for multiple testing, significant ( P < 0.05) changes in allele frequencies were detected by FET for 6 and 57 single nucleotide polymorphisms (SNPs) in the SR and RS populations, respectively, and by the CMH test for 124 SNPs in both populations. The significant variants located on chromosome 2 generated a selection signal in a genomic region harboring the mptl-1, deg-3 and des-2 genes, previously reported as candidates for monepantel resistance. In addition, three new variants were identified in the mptl-1 gene. Conclusions This study expands knowledge on genome-wide molecular events underlying H. contortus resistance to monepantel. The identification of a genome region harboring major genes previously associated with monepantel resistance supports the results of the employed X-QTL approach. In addition, a deletion in exon 11 of the mptl-1 gene should be further investigated as the putative causal mutation leading to monepantel resistance. Electronic supplementary material The online version of this article (10.1186/s13071-019-3663-9) contains supplementary material, which is available to authorized users.

Gastrointestinal nematodes significantly affect the ovine industry, and Haemonchus contortus is considered the most pathogenic parasite in tropical regions. This situation is aggravated when the main strategy to control worms fails because of the genetic resistance that parasites acquire against anthelmintics. Aiming to anticipate the events involved in anthelmintic resistance, we induced monepantel resistance in H. contortus by in vivo subdosing of sheep hosts. Four successive passages of a monepantel-susceptible H. contortus isolate in Santa Ines or Ile de France sheep hosts resulted in three monepantel-resistant (efficacy varying from 0 to 58.5%) H. contortus isolates. Sheep hosts were treated from 0.075 mg/kg to the therapeutic dose of 2.5 mg/kg of monepantel in 19–26 rounds of selection for 112–133 weeks. Success in inducing H. contortus resistance to monepantel may have been affected by worm burden and by host–parasite interactions, including a possible effect of the breed of sheep hosts. We conclude that subdosing of sheep, although time-consuming, is an efficient in vivo strategy for the induction of monepantel resistance in H. contortus. The resistant parasites can be used in further studies to elucidate the genetic and biochemical events involved in the acquisition of anthelmintic resistance.

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Single nucleotide polymorphisms (SNPs) are the main type of variation in genome, enabling them to be associated with traits of economic importance in livestock. Genome-wide association studies (GWAS) have led to the discovery of SNPs associated with desirable traits in sheep. However, in these studies, SNPs are genotyped by high-throughput methods in genome scale, which are expensive and require sophisticated equipment and analysis methods. Therefore, the goal of this study was to develop a reliable, rapid, and inexpensive polymerase chain reaction (PCR)-based method to genotype a medium number of animals for a few candidate SNPs previously associated with desirable phenotypes in sheep by GWAS, using markers associated with gastrointestinal nematode resistance as a model. DNA extracted from white-blood cells of 150 sheep was submitted to PCR amplification followed by agarose gel electrophoresis and determination of banding pattern. Tetra-primer ARMS-PCR was successfully optimized after changes in annealing temperature; annealing and extension times; concentration of MgCl and DNA; ratios of inner, outer, forward and reverse primer; and addition of adjuvants, for genotyping the OAR2_14765360, OAR6_81718546, OAR11_62887032, and OAR12_69606944 SNPs in sheep. An extensive optimization of tetra-primer ARMS-PCR resulted in a suitable, simple, cost-effective PCR-based method of genotyping four SNP markers previously detected by chip arrays.