title: Deep amplicon sequencing of preselected isolates of Parascaris equorum in β-tubulin codons associated with benzimidazole resistance in other nematodes.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  Two 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.
  
  
  RESULTS
  No 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Department of Biomedical Sciences and Veterinary Public Health, Division of Parasitology, Swedish University of Agricultural Sciences, Uppsala S-750 07, Sweden. Eva.Tyden@slu.se.
authors.forename: Eva
authors.initials: E
authors.lastname: Tydén
authors.forename: Johan
authors.initials: J
authors.lastname: Dahlberg
authors.forename: Olof
authors.initials: O
authors.lastname: Karlberg
authors.forename: Johan
authors.initials: J
authors.lastname: Höglund
mesh_terms: D000818:Animals; D000871:Anthelmintics / Q000494:pharmacology*; D001199:Ascaridoidea / Q000235:genetics / Q000378:metabolism*; D001562:Benzimidazoles / Q000494:pharmacology*; D003062:Codon; D004351:Drug Resistance; D005786:Gene Expression Regulation / Q000502:physiology; D021141:Nucleic Acid Amplification Techniques; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
chemical_list: D000871:Anthelmintics; D001562:Benzimidazoles; D003062:Codon; D014404:Tubulin; C031000:benzimidazole