Dataset Information

Large scale screening for benzimidazole resistance mutations in Nematodirus battus, using both pyrosequence genotyping and deep amplicon sequencing, indicates the early emergence of resistance on UK sheep farms.

ABSTRACT: Benzimidazoles (BZ) have been the anthelmintic of choice for controlling Nematodirus battus infections since their release in the 1950s. Despite heavy reliance on this single anthelmintic drug class, resistance was not identified in this nematode until 2010 (Mitchell et al., 2011). The study aimed to explore the prevalence of BZ-resistance mutations in N. battus from UK sheep flocks using deep amplicon sequencing and pyrosequencing platforms. Based on evidence from other gastrointestinal nematodes, resistance in N. battus is likely to be conferred by single nucleotide polymorphisms (SNP) within the ?-tubulin isotype 1 locus at codons 167, 198 and 200. Pyrosequencing and deep amplicon sequencing assays were designed to identify the F167Y (TTC to TAC), E198A (GAA to GCA) and F200Y (TTC to TAC) SNPs. Nematodirus battus populations from 253 independent farms were analysed by pyrosequencing; 174 farm populations were included in deep amplicon sequencing and 170 were analysed using both technologies. F200Y was the most prevalent SNP identified throughout the UK, in 12-27% of the populations tested depending on assay, at a low overall individual frequency of 2.2?±?0.6% (mean?±?SEM, based on pyrosequencing results). Four out of the five populations with high frequencies (>20%) of the F200Y mutation were located in NW England. The F167Y SNP was identified, for the first time in this species, in four of the populations tested at a low frequency (1.2%?±?0.01), indicating the early emergence of the mutation. E198A or E198L were not identified in any of the isolates. Results obtained were comparable between both techniques for F200Y (Lins' CCC, rc?=?0.96) with discrepancies being limited to populations with low frequencies. The recent emergence of resistance in this species will provide a unique opportunity to study the early stages of anthelmintic resistance within a natural setting and track its progress in the future.

SUBMITTER: Melville LA

PROVIDER: S-EPMC7132121 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Large scale screening for benzimidazole resistance mutations in Nematodirus battus, using both pyrosequence genotyping and deep amplicon sequencing, indicates the early emergence of resistance on UK sheep farms.

Melville Lynsey A LA Redman Elizabeth E Morrison Alison A AA Rebecca Chen Pai Chia PC Avramenko Russell R Mitchell Sian S Van Dijk Jan J Innocent Giles G Sargison Fiona F Aitken Catriona C Gilleard John S JS Bartley Dave J DJ

International journal for parasitology. Drugs and drug resistance 20200306

Benzimidazoles (BZ) have been the anthelmintic of choice for controlling Nematodirus battus infections since their release in the 1950s. Despite heavy reliance on this single anthelmintic drug class, resistance was not identified in this nematode until 2010 (Mitchell et al., 2011). The study aimed to explore the prevalence of BZ-resistance mutations in N. battus from UK sheep flocks using deep amplicon sequencing and pyrosequencing platforms. Based on evidence from other gastrointestinal nematod ...[more]

PMID: 32251964

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Project description: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.

Project description:BackgroundAnthelmintic resistance to benzimidazole has been detected in the canine hookworm, Ancylostoma caninum. Benzimidazole resistance is believed to have developed originally in greyhounds, but has also been detected in non-greyhound pet dogs. The aim of this study was to validate a probe-based allele-specific real-time PCR tests for the F167Y polymorphism on the β-tubulin isotype-1 gene and to determine the geographic distribution.MethodsAllele-specific real-time PCR tests were established and validated to detect the codon 167 polymorphism in the Ancylostoma caninum β-tubulin isotype-1gene. Additionally, real-time PCR tests were validated for Ancylostoma spp. and Uncinaria stenocephala. Two nucleic acid extraction protocols were validated including mechanical disruption of parasite structures in stool. The frequency of the F167Y single nucleotide polymorphism (SNP) was determined in hookworm confirmed stool samples. Samples with the resistant 167Y genotype were confirmed by β-tubulin gene sequencing and allele frequencies were determined.ResultsThe Ancylostoma spp. and A. caninum F167Y allele-specific real-time PCR tests were highly sensitive and specific when tested against synthetic DNA, spiked samples, and characterized parasites. Using an optimized total nucleic acid extraction protocol, 54 of 511 (10.6%) were found to contain the benzimidazole resistance allele. All 55 samples containing hookworms with the resistance mutation were confirmed by β-tubulin gene sequencing. The majority of resistant hookworms (44 resistant, 183 tested; 24.4%) originated from Florida, five from California (103 tested, 4.9%), three from Idaho (40 tested, 7.5%), two from Nevada (22 tested, 9.1%), and one sample from Hawaii (13 tested, 7.7%). Resistant genotypes were found in 14 different dog breeds including eight in Greyhounds. Allele-frequency determination revealed resistance allele frequencies between 1 and 100% with 58% above 50%.ConclusionsThis data strongly supports recent findings of benzimidazole resistant canine hookworms present throughout the general US pet dog population.

Project description:BackgroundThe 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.MethodsTwo 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.ResultsNo 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.ConclusionsWe 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.

Dataset Information

Large scale screening for benzimidazole resistance mutations in Nematodirus battus, using both pyrosequence genotyping and deep amplicon sequencing, indicates the early emergence of resistance on UK sheep farms.

Publications

Large scale screening for benzimidazole resistance mutations in Nematodirus battus, using both pyrosequence genotyping and deep amplicon sequencing, indicates the early emergence of resistance on UK sheep farms.

Similar Datasets

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