Deep amplicon sequencing of preselected isolates of Parascaris equorum in β-tubulin codons associated with benzimidazole resistance in other nematodes.

Eva Tydén,Olof Karlberg,Johan Höglund,Johan Dahlberg

doi:10.1186/1756-3305-7-410

Abstract

BackgroundThe development of anthelmintic resistance (AR) to macrocyclic lactones in the equine roundworm Parascaris equorum has resulted in benzimidazoles now being the most widely used substance to control Parascaris infections. However, over-reliance on one drug class is a risk factor for the development of AR. Consequently, benzimidazole resistance is widespread in several veterinary parasites, where it is associated with single nucleotide polymorphisms (SNPs) in drug targets encoded by the β-tubulin genes. The importance of these SNPs varies between different parasitic nematodes, but it has been hypothesised that they occur, at low allele frequencies, even in unselected populations. This study investigated whether these SNPs exist in the P. equorum population and tested the hypothesis that BZ resistance can develop from pre-existing SNPs in codons 167, 198 and 200 of the β-tubulin isotype 1 and 2 genes, reported to be associated with AR in strongylids. The efficacy of the oral paste formula fenbendazole on 11 farms in Sweden was also assessed.MethodsTwo isotype-specific primer pairs were designed, one on either side of the codon 167 and one on either side of codons 198 and 200. A pool of 100 000 larvae was sequenced using deep amplicon sequencing by Illumina HiSeq. Faecal egg count reduction test was used to assess the efficacy of fenbendazole.ResultsNo SNPs were observed in codons 167, 198 or 200 of the β-tubulin isotype 1 or 2 genes of P. equorum, even though 100 000 larvae were sequenced. Faecal egg count reduction testing of fenbendazole showed that this anthelmintic was still 100% effective, meaning that the likelihood of finding high allele frequency of SNPs associated with benzimidazoles resistance in P. equorum was low. Unexpectedly, the allele frequencies observed in single worms were comparable to those in pooled samples.ConclusionsWe concluded that fenbendazole does not exert selection pressure on the β-tubulin genes of isotypes 1 and 2 in P. equorum. The fact that no pre-existing SNPs were found in codons 167, 198 and 200 in P. equorum also illustrates the difficulties in generalising about AR mechanisms between different taxonomic groups of nematodes.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-3305-7-410) contains supplementary material, which is available to authorized users.

Highlights

The development of anthelmintic resistance (AR) to macrocyclic lactones in the equine roundworm Parascaris equorum has resulted in benzimidazoles being the most widely used substance to control Parascaris infections
Since the macrocyclic lactones (ML) ivermectin (IVM) was first introduced in the beginning of the 1980s many horses have been dewormed with this compound up to six times during their first year, which has resulted in the development of ML resistance in the P. equorum population [3]
Reliance on one drug class poses a threat of multiple AR developing in P. equorum, especially in light of recent findings on the genetic homogeneity of the global Parascaris population and the high gene flow within this population [36]

Summary

Introduction

The development of anthelmintic resistance (AR) to macrocyclic lactones in the equine roundworm Parascaris equorum has resulted in benzimidazoles being the most widely used substance to control Parascaris infections. Benzimidazole resistance is widespread in several veterinary parasites, where it is associated with single nucleotide polymorphisms (SNPs) in drug targets encoded by the β-tubulin genes The importance of these SNPs varies between different parasitic nematodes, but it has been hypothesised that they occur, at low allele frequencies, even in unselected populations. Parascaris equorum is a nematode parasite of foals and yearlings with a global distribution It has a direct life cycle and is one of the most important equine parasites because of its high prevalence and severe pathogenicity [1]. For many years, this ascarid roundworm could be controlled by strategic use of different anthelmintics, including macrocyclic lactones (ML), benzimidazoles (BZ) and tetrahydropyrimidines (TH) [2]. The BZs fenbendazole (FBZ) and oxibendazole (OBZ) are still effective and is the anthelmintic drug class currently recommended for treatment of P. equorum infection [6]

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