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. 2013 Aug 12;8(8):e70212.
doi: 10.1371/journal.pone.0070212. eCollection 2013.

Phylogenetic Characterization of β-Tubulins and Development of Pyrosequencing Assays for Benzimidazole Resistance in Cattle Nematodes

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Free PMC article

Phylogenetic Characterization of β-Tubulins and Development of Pyrosequencing Assays for Benzimidazole Resistance in Cattle Nematodes

Janina Demeler et al. PLoS One. .
Free PMC article

Abstract

Control of helminth infections is a major task in livestock production to prevent health constraints and economic losses. However, resistance to established anthelmintic substances already impedes effective anthelmintic treatment in many regions worldwide. Thus, there is an obvious need for sensitive and reliable methods to assess the resistance status of at least the most important nematode populations. Several single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene of various nematodes correlate with resistance to benzimidazoles (BZ), a major anthelmintic class. Here we describe the full-length β-tubulin isotype 1 and 2 and α-tubulin coding sequences of the cattle nematode Ostertagia ostertagi. Additionally, the Cooperia oncophora α-tubulin coding sequence was identified. Phylogenetic maximum-likelihood analysis revealed that both isotype 1 and 2 are orthologs to the Caenorhabditis elegans ben-1 gene which is also associated with BZ resistance upon mutation. In contrast, a Trichuris trichiura cDNA, postulated to be β-tubulin isotype 1 involved in BZ resistance in this human parasite, turned out to be closely related to C. elegans β-tubulins tbb-4 and mec-7 and would therefore represent the first non-ben-1-like β-tubulin to be under selection through treatment with BZs. A pyrosequencing assay was established to detect BZ resistance associated SNPs in β-tubulin isotype 1 codons 167, 198 and 200 of C. oncophora and O. ostertagi. PCR-fragments representing either of the two alleles were combined in defined ratios to evaluate the pyrosequencing assay. The correlation between the given and the measured allele frequencies of the respective SNPs was very high. Subsequently laboratory isolates and field populations with known resistance status were analyzed. With the exception of codon 167 in Cooperia, increases of resistance associated alleles were detected for all codons in at least one of the phenotypically resistant population. Pyrosequencing provides a fast, inexpensive and sensitive alternative to conventional resistance detection methods.

Conflict of interest statement

Competing Interests: As outlined in the financial disclosure section, the work described in the present manuscript was partially supported by Bayer Animal Health, Leverkusen. Bayer Animal Health (BAH) had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare that the funding through BAH does not alter their adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Phylogram showing relationship between nematode and D. melanogaster tubulins.
Deduced proteins were aligned and the corresponding alignment of cDNAs with aligned codons was analyzed by maximum likelihood analysis as described in material and methods. Statistical support according to the Shimodaira-Hasegawa modification of the approximate likelihood test and the Bayesian transformation of the approximate likelihood ratio are reported before and after the slash. If only one number is given the results of both tests were identical. Accession numbers for all cDNAs are provided in Table S2. Species abbreviations: Aca, Ancylostoma caninum; Alu, Ascaris lumbricoides; Asu, Ascaris suum; Bma, Brugia malayi; Can, Cylicocylus nassatus; Cbr, Caenorhabditis briggsae; Cca, Cylicocyclus catenatus; Cel, Caenorhabditis elegans; Con, Cooperia oncophora; Cpe, Cooperia pectinata; Dme, Drosophila melanogaster; Hco, Haemonchus contortus; Oos, Ostertagia ostertagia; Peq, Parascaris equorum; Tsp, Trichinella spiralis; Ttr, Trichuris trichiura. An enlarged version of the β-tubulin subtree is provided in Figure S1.
Figure 2
Figure 2. Regression analysis for pyrosequencing assays of C. oncophora β-tubulin isotype 1.
Artificial mixtures of respective PCR-products as templates containing varying amounts of copies with TAC in codon 168 (A), codon 198 (B) or codon 200 (C) were analyzed by pyrosequencing. Observed frequencies (mean ± SD) were plotted against input TAC frequencies in (A) and (C) and input GCA frequencies in (B). Regression plots (including linear equation and Pearson correlation coefficient) with 95% confidence bands are shown.
Figure 3
Figure 3. Regression analysis for pyrosequencing assays of O. ostertagi β-tubulin isotype 1.
Artificial mixtures of respective PCR-products as templates containing varying amounts of copies with TAC in codon 168 (A), codon 198 (B) or codon 200 (C) were analyzed by pyrosequencing. Observed frequencies (mean ± SD) were plotted against input TAC frequencies in (A) and (C) and input GCA frequencies in (B). Regression plots (including linear equation and Pearson correlation coefficient) with 95% confidence bands are shown.
Figure 4
Figure 4. Representative pyrograms for Cooperia oncophora and Ostertagia ostertagi β-tubulin isotype 1 SNPs.
Pyrograms show signal intensity on the ordinate and the dispersion pattern on the ordinate. Dispersions are abbreviated as follows: E, enzyme; S, substrate, A, dATP, C, dCTP, G, dGTP, T, dTTP.

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Grant support

The work presented herein was financed by the authors' resources and in part supported by an EU FP7-grant (GLOWORM, FP7-KBBE-2011-5 Area 2.1.3, KBBE 2011.1.3-04, Project-No. 288975) and Bayer Animal Health, Leverkusen, Germany. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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