Genetic variants in dyf-7 validated by droplet digital PCR are not drivers for ivermectin resistance in Haemonchus contortus

Abstract

Resistance to ivermectin (IVM) in the nematode Haemonchus contortus in small ruminants is an increasing problem throughout the world. Access to molecular diagnostics will allow early detection of IVM resistance, which in turn can limit the spread of resistant isolates. One candidate gene which has recently been suggested as a marker for IVM resistance is that for dye-filling protein (dyf-7). In this study, we critically investigated the suitability of A141G and G153T single nucleotide polymorphisms (SNP) of dyf-7 as a marker in larval cultures collected from sheep farms in Sweden, involving several isolates for which resistance status had been characterised by the faecal egg count reduction test (FECRT). Initially, we designed dyf-7 primers from a worldwide collection of adult Haemonchus contortus DNA. With the sequence data, we created a haplotype network. We then optimised and used the same sets of primers and probes in a droplet digital PCR (ddPCR) assay for precise quantification of dyf-7 allele frequencies in pre- and post-anthelmintic treatment faecal larval cultures. The fractional abundance (FA) of the mutant SNP was within the range 7.8 and 31%. However, the FA was generally stable in samples collected from the same farms, even though they were obtained on different occasions up to 25 months apart. There was also no indication that the level of IVM resistance as measured by the faecal egg count reduction test was higher on farms with high FA. Furthermore, by comparing FA in samples from the same farms pre- and post-IVM treatment, we found no evidence of a correlation between dyf-7 and level of IVM resistance. Based on these results, dyf-7 is not a suitable marker for field testing of IVM resistance in H. contortus.

Exploring benzimidazole resistance in Haemonchus contortus by next generation sequencing and droplet digital PCR

“…Thresholds, separating droplet clusters, were manually adjusted to obtain the best possible separation: Channel 1, which captures the fluorescence of the FAM™ dye molecule, attached to the wild type probe, had a threshold set at 7000 AU, whereas Channel 2, which respectively measures the fluorescence emitted by the HEX™ dye, attached to the mutant probe, at 5000 AU. A limit of detection (LoD) and fractional abundance (FA) precision identification in five wild-type (WT) and mutant (MT) mixtures (at 100/0, 75/25, 50/50, 25/75 and 0/100% ratios) tests (described by Elmahalawy et al, 2018 ) were carried out to evaluate the reliability of the designed assay.…”

Molecular detection of two major gastrointestinal parasite genera in cattle using a novel droplet digital PCR approach

“…The molecular techniques designed for either of the aforementioned purposes range from the very first few PCR-linked restriction fragment linked polymorphism assays (Gasser et al 1994) to semi-quantitative, real-time (Höglund et al 2013), and multiplex tandem (Roeber et al 2017) PCRs, as well as the recently introduced third-generation droplet digital (dd)PCR techniques (Baltrušis et al 2018; Elmahalawy et al 2018b). However, the majority of work and effort into improving the detection of nematode parasites in livestock go into the small ruminant sector, where the problem of drug-resistant worms is the most severe (Wolstenholme et al 2004).…”

Challenges and opportunities for the adoption of molecular diagnostics for anthelmintic resistance

“…Although some encouraging results have been reported, evidence of genetic associations between these candidates and phenotypic resistance have been generally inconsistent across studies ( Kotze et al, 2014 ). For example, a subset of SNPs within the dyf-7 gene common to multiple ivermectin-resistant isolates of H. contortus with sensory amphid neuron defects from five continents ( Urdaneta-Marquez et al, 2014 ) were subsequently shown to be not predictive of resistance in other H. contortus isolates from Africa, Australia and Europe ( Laing et al, 2016 ; Rezansoff et al, 2016 ; Elmahalawy et al, 2018 ). The recent transition from candidate-based single (or few) gene approaches to unbiased genome-wide and genetic mapping approaches has begun to resolve some of the genetic uncertainty associated with candidate genes, and in turn, identify promising new markers linked to resistance.…”