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 in 39 sheep flocks. Around 1,000 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 32% by FECRT or 26% by EHT, however F200Y and E198A SNPs were absent in T. circumcincta . The amplification 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 = 0.000) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4 and 80.7% before treatment, and from 82.3 to 92.8% after treatment. Very 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 = 0.000), 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 = 0.000). 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 .