Abstract
The ruminant livestock production sector is under threat due to the infections with gastrointestinal nematode parasites and the subsequent development of anthelmintic resistance. One of most common and pathogenic species in small ruminants is Haemonchus contortus. The ability to control the infections with this and other gastrointestinal nematodes relies heavily on the use of anthelmintic drugs. Although resistance to all major classes of anthelmintics has been shown in H. contortus, the precise mechanism of resistance acquisition is only known for benzimidazoles. F200Y (TAC) is a common point mutation in the isotype 1 β tubulin gene which is associated with an effective increase in the resistance towards benzimidazole drugs. Here, we show the utility of using this mutation as a marker in a droplet digital PCR assay to track how two H. contortus laboratory strains, characterized by different resistance levels, change with respect to this mutation, when subjected to increasing concentrations of thiabendazole. Additionally, we wanted to investigate whether exposure to a discriminating dose of thiabendazole in the egg hatch test resulted in the death of all H. contortus eggs with a susceptible genotype. We found the MHco5 strain to maintain an overall higher frequency of the F200Y mutation (80–100%) over all drug concentrations, whilst a steady, gradual increase from around 30%–60% was observed in the case of the MHco4 strain. This is further supported by the dose-response curves, displaying a much higher tolerance of the MHco5 strain (LD50 = 0.38 μg/ml) in comparison to the MHco4 strain (LD50 = 0.07 μg/ml) to the effects of thiabendazole. All things considered, we show that the F200Y mutation is still a viable and reliable marker for the detection and surveillance of benzimidazole drug resistance in H. contortus in Europe.
Highlights
Infections with gastrointestinal nematodes (GINs) in small rumi nants, pose an array of problems to the industry, mainly in the form of stunted growth reducing yields and the increasing costs of treatment
We utilized the previously developed ddPCR assay for F200Y mutation detection (Baltru sis et al, 2018), to estimate the fre quencies of the mutated (F200Y mutation possessing) β tubulin allele in the L1 (after the eggs had been hatched under gradually increasing thiabendazole (TBZ) concentrations) of two laboratory maintained strains of H. contortus, MHco4 and MHco5, exhibiting distinct resistance statuses
Droplet digital PCR was run on extracted DNA samples using a pre viously described set up for the identification of the fractional abun dance of the F200Y mutation in the isotype 1 β tubulin gene (Baltru sis et al, 2018)
Summary
Infections with gastrointestinal nematodes (GINs) in small rumi nants, pose an array of problems to the industry, mainly in the form of stunted growth reducing yields and the increasing costs of treatment. We utilized the previously developed ddPCR assay for F200Y mutation detection (Baltru sis et al, 2018), to estimate the fre quencies of the mutated (F200Y mutation possessing) β tubulin allele in the L1 (after the eggs had been hatched under gradually increasing thiabendazole (TBZ) concentrations) of two laboratory maintained strains of H. contortus, MHco and MHco, exhibiting distinct resistance statuses. Both strains have been previously shown to not be polymorphic for the 198 codon and only the MHco strain displayed low levels of the polymorphism in the 167 codon (7.2%) (von Samson-Himmeltjerna et al, 2009). The aim of this experimental study was to evaluate if the L1 of H. contortus, which hatched at doses higher than the discriminating (0.1 μg/ml) in the EHT possessed only the resistant ge notype (homozygous for F200Y) and overall describe the changes in the frequency of the mutant allele, for two differently resistant strains
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