Development of anthelminthic assays and characterization of the beta-tubulin gene family: Evaluating benzimidazole efficacy in Ascaridia galli

Since the ban on battery cage systems, infections with poultry nematode Ascaridia galli have increased dramatically. Benzimidazole (BZ) anthelminthic drugs are often used to treat these infections, but widespread use of anthelminthics can lead to the development of BZ resistance. BZ resistance is currently still rare in ascarids, while it is already widespread in strongyle nematodes. In these strongyle nematodes, BZ resistance is linked to single nucleotide polymorphisms (SNPs) at codons 167, 198 and 200 in the isotype 1 beta-tubulin gene. In the ascarids, very little is known about the beta-tubulin genes and their role in BZ resistance. Previously, only one beta-tubulin gene had been identified for A. galli. In this study, four additional beta-tubulin genes are presented, which have been identified from RNA-seq data, and analysed based on their sequences and expression patterns. Additionally, in vitro anthelminthic assays were performed to determine effectivity of BZs in a larval development assay (LDA) and larval migration inhibition assay (LMIA). These assays demonstrated efficacy of BZs, with albendazole having more efficacy in ovicidal activity, and fenbendazole in larvicidal activity. This suggests that BZ activity might vary across the life stages or that fenbendazole-resistant eggs are present, but further optimisation and field confirmation are necessary. Genetic analysis of the beta-tubulin genes identified 3 candidates for drug binding, in which no canonical resistance SNPs were found. This study suggests that different drug-binding sites are involved in ascarids and that resistance might involve alternative molecular mechanisms. The findings underscore the necessity of continued monitoring of BZ resistance and population-wide genetic screening to detect early resistance signs.