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

Abstract Background There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequencing, and standardized it for large-scale benzimidazole efficacy screening use. Methods Three different protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by flotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at different proportions, simulating different resistance levels. These mixtures were used to compare previously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defined, the utility of the protocols was assessed by measuring the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. Results The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequencing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample. Conclusions We optimized the sample processing methodology and standardized pyrosequencing in soil-transmitted helminth (STH) pooled eggs. These protocols could be used in STH large-scale screenings or anthelmintic efficacy trials. Graphical Abstract

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Assessing anthelmintic resistance risk in the post-genomic era: a proof-of-concept study assessing the potential for widespread benzimidazole-resistant gastrointestinal nematodes in North American cattle and bison

Parasitology ◽

10.1017/s0031182020000426 ◽

2020 ◽

Vol 147 (8) ◽

pp. 897-906 ◽

Cited By ~ 1

Author(s):

Russell W. Avramenko ◽

Elizabeth M. Redman ◽

Claire Windeyer ◽

John S. Gilleard

Keyword(s):

Drug Resistance ◽

Large Scale ◽

Anthelmintic Resistance ◽

Low Frequency ◽

Nematode Species ◽

Amplicon Sequencing ◽

Gastrointestinal Nematodes ◽

Genomic Research ◽

Resistance Risk ◽

Benzimidazole Resistance

AbstractAs genomic research continues to improve our understanding of the genetics of anthelmintic drug resistance, the revolution in DNA sequencing technologies will provide increasing opportunities for large-scale surveillance for the emergence of drug resistance. In most countries, parasite control in cattle and bison has mainly depended on pour-on macrocyclic lactone formulations resulting in widespread ivermectin resistance. Consequently, there is an increased interest in using benzimidazole drugs which have been used comparatively little in cattle and bison in recent years. This situation, together with our understanding of benzimidazole resistance genetics, provides a practical opportunity to use deep-amplicon sequencing to assess the risk of drug resistance emergence. In this paper, we use deep-amplicon sequencing to scan for those mutations in the isotype-1 β-tubulin gene previously associated with benzimidazole resistance in many trichostrongylid nematode species. We found that several of these mutations occur at low frequency in many cattle and bison parasite populations in North America, suggesting increased use of benzimidazole drugs in cattle has the potential to result in widespread emergence of resistance in multiple parasite species. This work illustrates a post-genomic approach to large-scale surveillance of early emergence of anthelmintic resistance in the field.

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Molecular basis for benzimidazole resistance from a novel β-tubulin binding site model

Journal of Molecular Graphics and Modelling ◽

10.1016/j.jmgm.2013.07.008 ◽

2013 ◽

Vol 45 ◽

pp. 26-37 ◽

Cited By ~ 33

Author(s):

Rodrigo Aguayo-Ortiz ◽

Oscar Méndez-Lucio ◽

Antonio Romo-Mancillas ◽

Rafael Castillo ◽

Lilián Yépez-Mulia ◽

...

Keyword(s):

Binding Site ◽

Molecular Basis ◽

Site Model ◽

Benzimidazole Resistance ◽

Tubulin Binding ◽

Β Tubulin

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First identification of the F200Y SNP in the β-tubulin gene linked to benzimidazole resistance in Ancylostoma caninum

Veterinary Parasitology ◽

10.1016/j.vetpar.2014.10.021 ◽

2014 ◽

Vol 206 (3-4) ◽

pp. 313-316 ◽

Cited By ~ 20

Author(s):

Luis Fernando Viana Furtado ◽

Ana Cristina Passos de Paiva Bello ◽

Hudson Andrade dos Santos ◽

Maria Raquel Santos Carvalho ◽

Élida Mara Leite Rabelo

Keyword(s):

Tubulin Gene ◽

Ancylostoma Caninum ◽

Benzimidazole Resistance ◽

Β Tubulin

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Multiple drug resistance in the canine hookworm Ancylostoma caninum: an emerging threat?

Parasites & Vectors ◽

10.1186/s13071-019-3828-6 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 17

Author(s):

Pablo D. Jimenez Castro ◽

Sue B. Howell ◽

John J. Schaefer ◽

Russell W. Avramenko ◽

John S. Gilleard ◽

...

Keyword(s):

Anthelmintic Resistance ◽

Multiple Drug Resistance ◽

Amplicon Sequencing ◽

Multiple Drug ◽

Tubulin Gene ◽

Ancylostoma Caninum ◽

The Usa ◽

Hookworm Infections ◽

Β Tubulin

Abstract Background The canine hookworm, Ancylostoma caninum is the most prevalent and important intestinal nematode parasite of dogs in the USA. Hookworms are typically well controlled by treatment with all commonly used anthelmintics that are approved for this use in dogs. However, in the past few years, cases of recurrent/persistent canine hookworm infections appear to have dramatically increased, suggesting that anthelmintic resistance (AR) may have evolved in this parasite. These cases are highly overrepresented by greyhounds, but multiple other breeds are also represented. The aim of this study was to characterize several of these suspected resistant isolates using in vitro, genetic and clinical testing to determine if these cases represent true anthelmintic resistance in A. caninum. Methods Fecal samples containing hookworm eggs from three cases of persistent hookworm infections; one from a greyhound, one from a miniature schnauzer and one from a hound-mix, were received by our laboratory. These were then used to establish infections in laboratory dogs and to perform egg hatch assays (EHA) and larval development assays (LDA) for detecting resistance to benzimidazoles and macrocyclic lactones, respectively. Additional EHA and LDA were performed on eggs recovered from the laboratory-induced infections. Fecal egg count reduction tests were performed to detect resistance to pyrantel. Deep amplicon sequencing assays were developed to measure the frequency of non-synonymous single nucleotide polymorphisms (SNP) at codons 167, 198 and 200 of the A. caninum isotype-1 β-tubulin gene. Results Resistance ratios for the three A. caninum isolates tested ranged from 6.0 to > 100 and 5.5 to 69.8 for the EHA and LDA, respectively. Following treatment with pyrantel, reduction in faecal egg counts was negative or 0%. Deep amplicon sequencing of the isotype-1 β-tubulin gene identified a high frequency of resistance-associated SNPs at codon 167 in all three resistant isolates and in two additional clinical cases. Conclusions These data conclusively demonstrate multiple anthelmintic resistance in multiple independent isolates of A. caninum, strongly suggesting that this is an emerging problem in the USA. Furthermore, evidence suggest that these resistant hookworms originate from racing greyhound farms and kennels, though additional research is needed to confirm this.

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