Utilization of droplet digital PCR to survey resistance associated polymorphisms in the β tubulin gene of Haemonchus contortus in sheep flocks in Sweden

Abstract

Infections with gastrointestinal nematodes (GINs) in small ruminants are becoming increasingly harder to treat due to the development of anthelmintic resistance. Across Swedish sheep farms, Haemonchus contortus is one of the more persistent and pathogenic species encountered. Benzimidazole drugs, such as albendazole, are still widely used to control the GIN burden in small ruminants. However, the decline in efficacy of this drug has been observed across the country. In this study, we aimed to continue to investigate the presence of single nucleotide polymorphisms in the β tubulin gene associated with benzimidazole drug resistance in H. contortus. This was carried out for sheep flocks from 67 farms around Sweden by screening for the two most commonly encountered SNPs at codons 167 and 200 in the isotype 1 β tubulin gene utilizing the droplet digital PCR technology. We first established a good agreement (Lin's concordance correlation coefficient = 0.987) between the previously widely used pyrosequencing assay for the detection of the SNP at codon 200 (otherwise known as mutation F200Y) and our assay, as well as developed and validated primer-probe pairs for the detection of the mutation at codon 167 (mutation F167Y) in the β tubulin gene of H. contortus. We then screened 174 pooled larval culture samples, collected either pre- or post-treatment, for the frequencies of the mutations F167Y and F200Y. Not only did we find the latter to be present at much higher frequencies, but the overall levels of this resistance conferring mutation have stayed stable throughout the years 2014-2019 at an average value of 88.5 ± 20.3% in the pre-treatment samples across the tested farms (p = 0.61, Kruskal-Wallis test). Furthermore, after establishing a mixed model and fitting our data, we found a significant (p < 0.01) difference in the average frequency of the mutation F200Y between paired, pre- and post-treatment with albendazole, samples. Although the frequency difference in samples treated with albendazole was relatively minor (88.5% in pre- and 95.6% in post-treatment), no significant (p = 0.15) change in F200Y mutation frequency was observed between the samples from the flocks treated with ivermectin (90.8% and 92.6 %, respectively).

Keywords: AR; Anthelmintic resistance; Benzimidazoles; F167Y; F200Y; Haemonchus contortus; ddPCR; isotype 1 β tubulin.