Benzimidazole resistance survey for Haemonchus , Teladorsagia and Trichostrongylus in three European countries using pyrosequencing including the development of new assays for Trichostrongylus

Abstract

Resistance to benzimidazoles (BZs) in trichostrongyloid nematodes is a worldwide problem for livestock production, particularly regarding small ruminants. Sensitive and reliable methods are required to assess anthelmintic resistance status. Currently available methods for BZ resistance detection can be divided into three main groups, in vivo (e.g. faecal egg count reduction test), in vitro (e.g. egg hatch assay) and molecular tests. Three single nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene of various nematode species correlate with BZ resistance. While PCR-based methods have been reported for the three most economically important nematodes of sheep, namely, Trichostrongylus, Haemonchus and Teladorsagia, pyrosequencing assays are so far only available for the latter two. Here, the design and evaluation of pyrosequencing assays for isotype-1 and isotype-2 β-tubulin genes of Trichostrongylus colubriformis are described. PCR fragments carrying the susceptible and corresponding resistant genotype were combined in defined ratios to evaluate assay sensitivity and linearity. The correlation between the given and the measured allele frequencies of the respective SNPs (codons F167Y, E198A and F200Y) was very high. Pyrosequencing assays for Haemonchus, Teladorsagia and Trichostrongylus were subsequently used for a BZ resistance survey, carried out in the three European countries, namely Ireland, Italy and Switzerland. Larval cultures obtained from field survey samples in 2012 and 2013 were used for pyrosequencing. The test was applied when the target species represented at least 10% of the sample. Trichostrongylus and Teladorsagia were detected in all countries' samples whereas Haemonchus was not detected in samples from Ireland. SNPs in isotype-1 associated with resistance were detected for all three species, with frequencies at codon F200Y far exceeding those at codons F167Y and E198A. Elevated SNP frequencies in isotype-2 of Trichostrongylus were only rarely detected. Farms with BZ resistance-associated SNP frequencies above 10% were most often found in Switzerland followed by Ireland and Italy.

New codon 198 β-tubulin polymorphisms in highly benzimidazole resistant Haemonchus contortus from goats in three different states in Sudan

“…The recently described codon 198 mutation (E198L) in T. circumcincta was also shown to be widespread at high frequency on many UK farms in Trichostrongylus axei [19,20]. Multiple molecular tools to qualitatively identify or quantify these variants have been developed [20,[24][25][26][27][28]. The present study aimed to understand the BZ-resistance mechanisms in H. contortus isolates from Sudan using pyrosequencing assays.…”

“…Molecular techniques have been established for qualitative detection and quantification of allele frequencies for the three substitutions F167Y, E198A and F200Y using conventional PCR, real time PCR, pyrosequencing assays, droplet digital PCR and deep amplicon sequencing in various parasitic nematodes including H. contortus, Trichostrongylus colubriformis, Cooperia oncophora, Ostertagia ostertagi and T. circumcincta [20,[24][25][26][27][28]. Pyrosequencing assays are now widely used to detect resistance predicting alleles in DNA extracted from field samples using pooled adult worms or larval stages [26,29,30].…”

Multiple Drug Resistance in the canine hookwormAncylostoma caninum: an Emerging Threat

“…Single nucleotide polymorphisms in the isotype-1 β-tubulin gene located at codons 167(TTC/Phe → TAC/Tyr), 198GAG/Glu → GCG/Ala) and 200(TTC/Phe → TAC/Tyr) are associated with benzimidazole resistance in multiple species of parasitic nematodes such as Haemonchus contortus (Kwa et al, 1994), Teladorsagia circumcincta (Elard et al, 1996) and cyathostomins (von Samson-Himmelstjerna et al, 2001). Several PCR and pyrosequencing assays have been developed to detect and measure these mutations, (Álvarez-Sánchez et al, 2005; Chaudhry et al, 2014; Demeler et al, 2013; Knapp-Lawitzke et al, 2015; Ramünke et al, 2016; Redman et al, 2015; von Samson-Himmelstjerna et al, 2009b) but these all have limitations that affect their usefulness.…”

High frequency of benzimidazole resistance alleles in trichostrongyloids from Austrian sheep flocks in an alpine transhumance management system

“…By contrast, molecular tests have very high sensitivity and specificity for the early detection of non-synonymous single nucleotide polymorphisms (SNPs), which are useful genetic markers for resistance [24,25]. Knowledge of the role of these SNPs and other genetic markers is only sufficiently characterized for BZs, so BZs are currently the only drug class where a SNP analysis can indicate a resistant phenotype.…”