Abstract
Anthelmintic resistant gastrointestinal helminths have become a major cause of poor health in sheep and goats. Sensitive and specific molecular markers are needed to monitor the genotypic frequency of resistance in field parasite populations. Gastrointestinal nematode resistance to benzimidazole is caused by a mutation in one of three positions within the isotype 1 β-tubulin gene. In the absence of markers for resistance to the other broad spectrum anthelmintic classes, these provide a relevant study example. Determination of the prevalence of these single nucleotide polymorphisms in field nematode populations can be impractical using conventional molecular methods to examine individual parasites; which can be laborious and lack sensitivity in determining low levels of resistance in parasite populations. Here, we report the development of a novel method based on an Illumina MiSeq deep amplicon sequencing platform to sequence the isotype 1 β-tubulin locus of the small ruminant gastrointestinal nematode, Teladorsagia circumcincta, and determine the frequency of the benzimidazole resistance mutations. We validated the method by assessing sequence representation bias, comparing the results of Illumina MiSeq and pyrosequencing, and applying the method to populations containing known proportions of resistant and susceptible larvae. We applied the method to field samples collected from ewes and lambs on over a period of one year on three farms, each highlighting different aspects of sheep management and approaches to parasite control. The results show opportunities to build hypotheses with reference to selection pressures leading to differences in resistance allele frequencies between sampling dates, farms and ewes or lambs, and to consider the impact of their genetic fixation or otherwise. This study provides proof of concept of a practical, accurate, sensitive and scalable method to determine frequency of anthelmintic resistance mutations in gastrointestinal nematodes in field studies and as a management tool for livestock farmers.
Highlights
Resistance to broad spectrum anthelmintic drugs is a global threat to efficient livestock production (Kaplan, 2004; Kaplan and Vidyashankar, 2012)
The F200Y (TAC) single nucleotide polymorphism (SNP) was found at different frequencies of between 67.2% and 94.5% in the four phenotypically benzimidazole resistant (2-R, 3-R, 5-R and 6-R) populations and consistently between 3% and 11.6% in the two phenotypically benzimidazole susceptible (1-S and 4-S) populations (Fig. 1 and Supplementary Table S3)
The benzimidazole resistance-associated F167Y (TAC) SNP was identified at a frequency of between 2% and 4.3% in the 6-R population (Fig. 1 and Supplementary Table S3)
Summary
Resistance to broad spectrum anthelmintic drugs is a global threat to efficient livestock production (Kaplan, 2004; Kaplan and Vidyashankar, 2012). Anthelmintic drugs belonging to the tubulin binding benzimidazole class are the mainstay for the control gastrointestinal nematode in lower and middle income countries (Ali et al, 2019; Chaudhry et al, 2015). Benzimidazole resistance is associated with mutations in the isotype 1 β-tubulin gene that prevent drug binding (Geary et al, 1992a). A single nucleotide polymorphism (SNP) mutation was first identified in IJP: Drugs and Drug Resistance 10 (2019) 92–100 benzimidazole resistant Haemonchus contortus and Trichostrongylus colubriformis resulting in an amino acid substitution from tyrosine to phenylalanine at position 200 (F200Y) of the polypeptide encoded by the isotype 1 β-tubulin gene (Kwa et al, 1994, 1995). A third isotype 1 β-tubulin mutation (E198A) with substitution from glutamate to alanine at position 198 was first identified in field populations of H. contortus (Chaudhry et al, 2015; Ghisi et al, 2007; Rufener et al, 2009) and has subsequently been reported in T. circumcincta with two substitutions (E198L [TTA] or E198A [GCA]) (Redman et al, 2015)
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