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Parasites & vectors2014; 7; 410; doi: 10.1186/1756-3305-7-410

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

Abstract: 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.
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Publication Date: 2014-08-29 PubMed ID: 25175357PubMed Central: PMC4156605DOI: 10.1186/1756-3305-7-410Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The article investigates the existence and impact of single nucleotide polymorphisms (SNPs) in the β-tubulin genes of the equine roundworm Parascaris equorum, associated with resistance to benzimidazoles, a common treatment for the infection. They found no pre-existing SNPs in key codons of these genes, implying that the treatment does not apply selection pressure that could lead to benzimidazole resistance in P. equorum.

Study Background and Purpose

  • In this research, the authors aimed to scrutinize whether SNPs responsible for resistance to benzimidazoles exist in the β-tubulin genes of the roundworm P. equorum. Benzimidazoles is prominently used for worm control because the worm develops resistance to other anthelmintics (anti-parasitic drugs).
  • The researchers hypothesized that SNPs within specific codons (167, 198, and 200) of the β-tubulin genes are present at low frequencies within P. equorum populations, even in non-resistant worms, which could potentially be the origin of benzimidazole resistance.

Methodology

  • The team created two primer pairs specific for different isotypes of the β-tubulin genes. One primer pair was specific for either side of codon 167, and the other for either side of codons 198 and 200.
  • They then used deep amplicon sequencing — an approach to sequence particular areas of interest within the genome — on a large sample of 100,000 larvae.
  • To further assess benzimidazoles (specifically, fenbendazole) efficacy, the faecal egg count reduction test was used among 11 Swedish farms.

Results and Conclusions

  • No SNPs were discovered in the β-tubulin genes of P. equorum, in codons 167, 198, or 200. This suggests that benzimidazoles do not apply selection pressure on these genes.
  • The faecal egg count reduction test revealed that fenbendazole continues to be 100% effective, implying a low likelihood of discovering high-frequency SNPs associated with benzimidazole resistance in P. equorum.
  • The lack of SNPs demonstrates difficulties in generalizing anthelmintic resistance mechanisms across different nematode groups.
  • Unexpectedly, allele frequencies in individual worms were comparable to those in pooled samples, which could provide insights for future research in sample analysis protocol.

Cite This Article

APA
Tydén E, Dahlberg J, Karlberg O, Höglund J. (2014). Deep amplicon sequencing of preselected isolates of Parascaris equorum in β-tubulin codons associated with benzimidazole resistance in other nematodes. Parasit Vectors, 7, 410. https://doi.org/10.1186/1756-3305-7-410

Publication

Parasites & vectors
ISSN: 1756-3305
NlmUniqueID: 101462774
Country: England
Language: English
Volume: 7
Pages: 410

Researcher Affiliations

Tydén, Eva
  • 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.
Dahlberg, Johan
    Karlberg, Olof
      Höglund, Johan

        MeSH Terms

        • Animals
        • Anthelmintics / pharmacology
        • Ascaridoidea / genetics
        • Ascaridoidea / metabolism
        • Benzimidazoles / pharmacology
        • Codon
        • Drug Resistance
        • Gene Expression Regulation / physiology
        • Nucleic Acid Amplification Techniques
        • Tubulin / genetics
        • Tubulin / metabolism

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        This article has been cited 8 times.
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