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

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.

Benzimidazole resistance survey for Haemonchus , Teladorsagia and Trichostrongylus in three European countries using pyrosequencing including the development of new assays for Trichostrongylus

“…Recent analyses showed no clear preference for anyof the three SNPs (Demeler et al., 2013, AlGusbi et al., 2014), although it is noteworthy that, in total, a much lower number of populations/isolates has so far been analysed in cattle parasitic nematodes compared to sheep.…”

“…There have been very few molecular genotyping studies performed in cattle parasitic nematodes. SNPs at all three codons have been associated with benzimidazole resistance in trichostrongylid parasite species of small ruminants and have been recently reported in three cattle parasites, H. placei , O. ostertagi and C. oncophora (Njue and Prichard, 2003, Winterrowd et al., 2003, Brasil et al., 2012, Demeler et al., 2013, Chaudhry et al., 2014). This diversity complicates the molecular detection and accordingly requires the examination of all three codons when molecular tests are applied to field samples.…”

“…The LDA is very sensitive but more labour-intensive than the EHA and also takes at least a week to provide results. Molecular tests include conventional PCR (Silvestre and Humbert, 2000, Njue and Prichard, 2003, Winterrowd et al., 2003), real-time PCR (Alvarez-Sanchez et al., 2005, Walsh et al., 2007) as well as pyrosequencing (Höglund et al., 2009, von Samson-Himmelstjerna et al., 2009, Demeler et al., 2013) and are all based on the detection of SNPs in the three above-mentioned codons in the isotype-1 β-tubulin gene.…”

Plant-Based Solutions to Global Livestock Anthelmintic Resistance

“…When feasible, using the latter method will provide more accurate, reproducible results. Benzimidazoles synthe c chemical α-and β-tubulin monomers (Demeler et al 2013) Dasa nib synthe c chemical protein kinases (Taylor et al 2013)…”

Molecular Diagnostics for Soil-Transmitted Helminths

“…are notorious for being more difficult to break open in the DNA extraction process than any other STH. Indeed, it is clear that a tissue homogenization step is imperative to achieve egg disruption 71. This parasite is the least well studied in the context of molecularly based diagnosis.…”