Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance

Abstract

Background Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta. Methods The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. Results The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4–80.7% before treatment, and 82.3–92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. Conclusions The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

Absence of Polymorphisms in Codons 167, 198 and 200 of All Seven β-Tubulin Isotypes of Benzimidazole Susceptible and Resistant Parascaris spp. Specimens from Australia

“…Initially, the amino acid exchanges F200Y [ 21 , 22 ], F167Y [ 23 , 24 ] and sometimes E198A [ 25 ] were predominantly reported in many resistant parasite populations. Recently, additional exchanges in codons 198, in particular E198L and E198V in resistant strongyle nematodes, have raised attention at least in some geographical regions [ 26 , 27 , 28 , 29 , 30 , 31 ]. Based on the knowledge that these mutations can confer resistance to BZs, epidemiological surveys have been established to rapidly identify BZ-resistant parasite populations, using methods such as pyrosequencing [ 26 , 27 , 28 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ], droplet digital PCR [ 31 , 40 ] or deep amplicon sequencing [ 29 , 41 ].…”

Susceptible trichostrongyloid species mask presence of benzimidazole-resistant Haemonchus contortus in cattle

“…In a very recent study from Spain, the frequency of E198L (GAA/CTA) was found to be highly associated with the response to BZ treatment in sheep naturally infected with T. circumcincta . In this study, the frequency of leucine was changed from the range of 10.4–80.7% in samples before treatment to a range of 82.3–92.8% after treatment [ 53 ]. In a very important new publication, leucine, alanine and valine were introduced at codon position 198 of the ben-1 β-tubulin isotype into the BZ susceptible, free-living Bristol N2 Caenorhabditis elegans using CRISPR/Cas-9 mediated genome editing followed by exposure to different concentrations of albendazole and fenbendazole.…”

Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs

“…The SNP F200Y has been shown to be strongly associated with low efficacy of benzimidazoles, whereas the SNPs F167Y and E198A have been occasionally related with anthelmintic resistance [ 28 ]. Besides that, the presence of the E198L polymorphism on its own, a substitution of glutamate to leucine, was very recently related to benzimidazole resistance in H. contortus and T. circumcincta , two gastrointestinal nematodes infecting ruminants [ 19 , 29 ]. In humans, only SNP F200Y has been associated with low efficacy of benzimidazoles in T. trichiura in one study [ 10 ], since the authors observed an increment in the resistant genotype in individual eggs collected after treatment.…”