Deep amplicon sequencing as a powerful new tool to screen for sequence polymorphisms associated with anthelmintic resistance in parasitic nematode populations

Avramenko, Russell W.; Redman, Elizabeth; Melville, Lynsey; Bartley, Yvonne; Wit, Janneke; Queiroz, Camila; Bartley, Dave; Gilleard, John S.

doi:10.1016/j.ijpara.2018.10.005

Cited by 96 publications

(156 citation statements)

References 64 publications

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“…Deep amplicon sequencing assays were developed to determine the frequency of non-synonymous single nucleotide polymorphisms at codons 167, 198 and 200 of the A. caninum isotype-1 β-tubulin gene. The approach and methods were as previously described for ruminant trichostrongylid nematodes except for the primer design (Avramenko et al, 2019). The presence of a large intron between exons 4 and 5 (1217 bp in reference sequence DQ459314.1 (GenBank accession)) meant that a single amplicon encompassing the three codons of interest would be too long for reliable Illumina sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…Consequently, primers were designed to amplify two separate regions of the A. caninum isotype-1 β-tubulin gene; a 293 bp fragment between exons 3 and 4 encompassing codon 167 and a 340 bp fragment between exons 5 and 6 encompassing codons 198 and 200 (Table 1). Using these primers, adapted primers suitable for Illumina next-generation sequencing were designed as previously described (Avramenko et al, 2019). The following PCR conditions were used to generate both fragments appropriate for sequencing: 5□μL of 5× NEB Q5 Reaction Buffer (New England Biolabs Ltd, USA) 0.5□μl.…”

Section: Methodsmentioning

confidence: 99%

“…The thermocycling parameters were 98□°C for 30□s, followed by 45 cycles of 98□°C for 10□s, 65□°C for 15□s, and 72□°C for 25□s, followed by 72°C for 2 min. Samples were purified and barcoded primers added following the protocols outlined in Avramenko et al , 2019 (Avramenko et al, 2019). Library preparation was as previously described and library sequencing performed using the Illumina MiSeq platform with the 2×250 v2 Reagent Kit (Illumina Inc., San Diego, CA, USA) (Avramenko et al, 2015).…”

Section: Methodsmentioning

confidence: 99%

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Multiple Drug Resistance in the canine hookwormAncylostoma caninum: an Emerging Threat

Castro

Howell

Schaefer

et al. 2019

Preprint

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In the past few years, diagnoses by veterinarians of recurrent canine hookworm infections have dramatically increased, suggesting that anthelmintic resistance (AR) may have evolved in the parasite Ancylostoma caninum. To investigate this, we established three “suspected-resistant” and two susceptible A. caninum isolates in research dogs for further study. The egg hatch assay (EHA) and the larval development assay (LDA) were used for detecting resistance to benzimidazoles, and macrocyclic lactones, respectively. Resistance ratios ranged from 6.0 to >100 and 5.5-69.8 for the EHA and LDA, respectively. Following treatments with fenbendazole, pyrantel and milbemycin oxime, reduction in faecal egg counts ranged from 64–86%, 0–72% and 58–92%, respectively. Deep amplicon sequencing of the isotype-1 β tubulin gene identified a high frequency of resistance-associated single nucleotide polymorphisms at codon 167 in the resistant isolates and clinical cases.. These data conclusively demonstrate multiple anthelmintic resistance in A. caninum, and provide pivotal evidence that this is an emerging problem in the United States. Consequently, these findings should provide some concern to the global health community, as the scale-up of mass drug administration for soil-transmitted helminths (STH) is now placing similar selection pressures for benzimidazole resistance in human hookworms.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Multiple Drug Resistance in the canine hookwormAncylostoma caninum: an Emerging Threat

Castro

Howell

Schaefer

et al. 2019

Preprint

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“…We have demonstrated the feasibility and practicality of deep amplicon sequencing by Illumina Mi-seq in this regard, using benzimidazole resistance in T. circumcincta as proof of concept, and have considered how knowledge of resistance allele frequencies in field populations might be used to build hypotheses on selection pressures and inform mitigation strategies. The method could be applied to other gastrointestinal nematode species, and multiplexed to study benzimidazole resistance co-infections (Avramenko et al, 2019). Once markers become available, the method might also be used to study single or multiple mutations conferring resistance to other anthelmintic drug groups, and combined with phylogenetic tools to show their emergence and spread.…”

Section: Discussionmentioning

confidence: 99%

Development of high throughput method for the analysis of anthelmintic resistance allele frequencies in field populations of gastrointestinal nematodes

MacLeay

et al. 2019

Preprint

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Drug resistant helminths have become a major cause of poor health and production in sheep and goats, and there is a need for diagnostic markers and tools to determine the frequency of resistance alleles in field parasite populations. Gastrointestinal nematode resistance to benzimidazole drugs is caused by a mutation in one of three positions on the isotype 1 β-tubulin locus, and in the absence of markers for resistance to other broad spectrum anthelmintic classes, these provide a relevant study example. Determination of the prevalence of these single nucleotide polymorphisms in field gastrointestinal nematode populations can be impractical using conventional molecular methods, which may be error prone or lack sensitivity at low levels of resistance. Here, we report the development of a novel method based on an Illumina Mi-seq 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 in the isotype 1 β-tubulin locus, comparing the results of Illumina Mi-seq and pyrosequencing, and applying the method to populations containing known proportions of resistant and susceptible L3. Finally, 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 drug resistance mutations in gastrointestinal nematodes in field studies and as a management tool for livestock farmers.

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“…Genomic approaches offer an information-rich technology for diagnostic and surveillance applications. Increasing throughput and decreasing costs of whole genome sequencing has resulted in the recent and steadily growing application of genomics in helminth parasitology; for example, for diagnostic applications, high throughput amplicon sequencing for helminth species identification and community composition (Avramenko et al, 2015) and the presence of drug resistance alleles (Avramenko et al, 2019) (Doyle et al, 2018) and microfilaria of Wuchereria bancrofti (Small et al, 2018) . Whole genome amplification protocols do, however, add considerable expense per sample, and can introduce technical artefacts such as uneven and/or preferential amplification (potentially of contaminant sequences), chimeric sequences, and allele dropout (Sabina and Leamon, 2015;Tsai et al, 2014) , that may lead to a reduction in genetic diversity, and in turn, relevance to the original unamplified material.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of DNA extraction methods on individual helminth egg and larval stages for whole genome sequencing

Doyle

Sankaranarayan

Allen

et al. 2019

Preprint

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Whole genome sequencing is being rapidly applied to the study of helminth genomes, including de novo genome assembly, population genetics, and diagnostic applications. Although late-stage juvenile and adult parasites typically produce sufficient DNA for molecular analyses, these parasitic stages are almost always inaccessible in the live host; immature life stages found in the environment for which samples can be collected non-invasively offer a potential alternative, however, these samples are typically yield very low quantities of DNA, can be environmentally resistant, and are susceptible to contamination, often from bacterial or host DNA. Here, we have tested five low-input DNA extraction protocols together with a low-input sequencing library protocol to assess the feasibility of whole genome sequencing of individual immature helminth samples. These approaches do not use whole genome amplification, a common but costly approach to increase the yield of low input samples. We first tested individual parasites from two species spotted onto FTA cards - egg and L1 stages of Haemonchus contortus and miracidia of Schistosoma mansoni - before further testing on an additional six species - Ancylostoma caninum, Ascaridia dissimilis, Dirofilaria immitis, Dracunculus medinensis, Strongyloides stercoralis, and Trichuris muris - with an optimal protocol. Whole genome sequencing followed by analyses to determine the proportion of on- and off-target mapping revealed successful sample preparations for six of the eight species tested with variation between species, and within species but between life stages, described. These results demonstrate the feasibility of whole genome sequencing of individual parasites, and highlight a new avenue towards generating sensitive, specific, and information-rich data for the diagnosis and surveillance of helminths.

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Deep amplicon sequencing as a powerful new tool to screen for sequence polymorphisms associated with anthelmintic resistance in parasitic nematode populations

Cited by 96 publications

References 64 publications

Multiple Drug Resistance in the canine hookwormAncylostoma caninum: an Emerging Threat

Multiple Drug Resistance in the canine hookwormAncylostoma caninum: an Emerging Threat

Development of high throughput method for the analysis of anthelmintic resistance allele frequencies in field populations of gastrointestinal nematodes

Evaluation of DNA extraction methods on individual helminth egg and larval stages for whole genome sequencing

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