Exploring the β-tubulin gene family in a benzimidazole-resistant Parascaris univalens population

Abstract

Benzimidazole (BZ) drugs are frequently used to treat infections with the equine ascarid Parascaris univalens due to increasing resistance to macrocyclic lactones and pyrantel. Benzimidazole resistance is rare in ascarids in contrast to strongyle parasites where this resistance is widespread. In strongyles, single nucleotide polymorphisms (SNPs) at codons 167, 198 and 200 in a β-tubulin gene have been correlated to BZ resistance, but little is known about the β-tubulin genes and their possible involvement in BZ resistance in P. univalens and other ascarids. Previously two β-tubulin genes have been identified in P. univalens . In this study, we present five additional β-tubulin genes as well as the phylogenetic relationship of all seven genes to β-tubulins of other clade III and V nematodes. In addition, the efficacy of fenbendazole for treatment of P. univalens on a Swedish stud farm was studied in 2019 and 2020 using faecal egg count reduction test. Reductions varied from 73% to 88%, indicating the presence of a resistant P. univalens population on the farm. The emergence of BZ resistance emphasizes the need for development of molecular markers for rapid and more sensitive detection of resistant populations. We therefore investigated whether possible SNPs at positions 167, 198 or 200 in any of the β-tubulin genes could be used to distinguish between resistant and susceptible P. univalens populations. Amplicon sequencing covering the mutation sites 167, 198 and 200 in all seven β-tubulin genes revealed an absence of SNPs in both resistant and susceptible populations, suggesting that the mechanism behind BZ resistance in ascarids is different from that in strongyle nematodes and the search for a molecular marker for BZ resistance in P. univalens needs to continue.

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

“…Resistance against macrocyclic lactones, in particular ivermectin, has been reported from Europe, North America, Asia and Australia [ 10 , 12 ]. Resistance to pyrantel [ 2 , 13 ] and benzimidazoles (BZ) [ 14 , 15 , 16 ] has also been reported occasionally. Regarding the pattern of resistance to drug classes, the situation in Parascaris spp.…”

“…Deep amplicon sequencing for the two most widely and highly expressed Ascaris β-tubulin genes revealed no evidence for SNPs associated with BZ resistance [ 43 ]. The β-tubulin repertoire of P. univalens has recently been characterised by Martin et al [ 16 ]. The authors identified a very closely related P. univalens ortholog to almost every Ascaris spp.…”

“…Β-tubulin isotype. With the exception of the tbb-B’ gene, which was only identified in the A. lumbricoides and not the A. suum reference genome by Roose et al [ 43 ], no P. univalens ortholog was detected [ 16 ]. Deep amplicon sequencing for all seven P. univalens β-tubulin paralogs confirmed the presence of tyrosine at codon 200 of the tbb-D gene but no polymorphisms in any of the other codons/isotypes.…”

“…Deep amplicon sequencing for all seven P. univalens β-tubulin paralogs confirmed the presence of tyrosine at codon 200 of the tbb-D gene but no polymorphisms in any of the other codons/isotypes. Malekpour et al [ 16 ] also found no polymorphisms in bt-A sequences obtained from Parascaris spp. Collected in Iran.…”

“…Although recent publications have characterised the β-tubulin genes of A. suum , A. lumbricoides [ 43 ] and P. univalens [ 16 ], none of these studies contained the complete β-tubulin repertoire of all three ascarid species in their phylogenetic analyses. In all three species, single orthologs of C. elegans mec-7 and tbb-4 genes were identified.…”