A repeatable and quantitative DNA metabarcoding assay to characterize mixed strongyle infections in horses

Poissant, Jocelyn; Gavriliuc, Stefan; Bellaw, Jennifer L.; Redman, Elizabeth; Avramenko, Russell W.; Robinson, David; Workentine, Matthew L.; Shury, Todd; Jenkins, Emily J.; McLoughlin, Philip D.; Nielsen, Martin K.; Gilleard, John S.

doi:10.1016/j.ijpara.2020.09.003

Cited by 47 publications

(47 citation statements)

References 44 publications

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“…The nematode communities identified from faecal samples using molecular and morphological methods were largely congruent: species identified from parasitological investigations were also recovered with the metabarcoding approach. This has been observed for other species, including horses and gorillas [ 32 , 54 , 55 ]. However, it must be noted that false negatives were detected for three species in the data set where forward and reverse reads were merged.…”

Section: Discussionsupporting

confidence: 65%

“…The identification of false negatives in the data that can be directly attributed to bioinformatic decisions highlights the need for consideration of a priori knowledge of the target organisms in order to optimize the metabarcoding methodology for these types of assays. Compared to conventional methods using morphological identification of adult worms, larvae, and eggs, molecular methods using faeces have higher throughput, are repeatable, provide species-level identifications of all GIN life stages, and have no dependence on lethal sampling [14,31,54]. Although a linear scaling between metabarcoding sequence abundance and larval abundance in faecal samples has been documented in several cases [31,55], we observe no correlation between egg counts and the proportion of nematode reads recovered, although it must be noted that our observations are based on only five individuals.…”

Section: Discussionmentioning

confidence: 99%

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Characterizing parasitic nematode faunas in faeces and soil using DNA metabarcoding

2021

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Background Gastrointestinal parasitic nematodes can impact fecundity, development, behaviour, and survival in wild vertebrate populations. Conventional monitoring of gastrointestinal parasitic nematodes in wild populations involves morphological identification of eggs, larvae, and adults from faeces or intestinal samples. Adult worms are typically required for species-level identification, meaning intestinal material from dead animals is needed to characterize the nematode community with high taxonomic resolution. DNA metabarcoding of environmental samples is increasingly used for time- and cost-effective, high-throughput biodiversity monitoring of small-bodied organisms, including parasite communities. Here, we evaluate the potential of DNA metabarcoding of faeces and soil samples for non-invasive monitoring of gastrointestinal parasitic nematode communities in a wild ruminant population. Methods Faeces and intestines were collected from a population of wild reindeer, and soil was collected both from areas showing signs of animal congregation, as well as areas with no signs of animal activity. Gastrointestinal parasitic nematode faunas were characterized using traditional morphological methods that involve flotation and sedimentation steps to concentrate nematode biomass, as well as using DNA metabarcoding. DNA metabarcoding was conducted on bulk samples, in addition to samples having undergone sedimentation and flotation treatments. Results DNA metabarcoding and morphological approaches were largely congruent, recovering similar nematode faunas from all samples. However, metabarcoding provided higher-resolution taxonomic data than morphological identification in both faeces and soil samples. Although concentration of nematode biomass by sedimentation or flotation prior to DNA metabarcoding reduced non-target amplification and increased the diversity of sequence variants recovered from each sample, the pretreatments did not improve species detection rates in soil and faeces samples. Conclusions DNA metabarcoding of bulk faeces samples is a non-invasive, time- and cost-effective method for assessing parasitic nematode populations that provides data with comparable taxonomic resolution to morphological methods that depend on parasitological investigations of dead animals. The successful detection of parasitic gastrointestinal nematodes from soils demonstrates the utility of this approach for mapping distribution and occurrences of the free-living stages of gastrointestinal parasitic nematodes. Graphical abstract

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Section: Discussionsupporting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Characterizing parasitic nematode faunas in faeces and soil using DNA metabarcoding

2021

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“…The ITS-2 rDNA region has been implemented already for characterizing equine strongylid communities (Poissant et al, 2021) but its predictive performances have not been characterized against mock equine strongylid communities. Its amplification was more robust as already reported (Louro et al, 2021) and the PCR amplification efficiency was consistent across considered species.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to this higher resolutive power, the protein-coding nature of the COI barcode can be leveraged to denoise sequencing data (Ramirez-Gonzalez et al, 2013). To date, metabarcoding experiments on equine strongylid species have exclusively focused on the ITS-2 rDNA cistron (Poissant et al, 2021). This may owe to the existence of universal primers and the length of the amplicon that is a good fit for short-read sequencing platforms.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the nemabiome approach for the study of equine strongylid communities

Courtot

Boisseau

Dhorne–Pollet

et al. 2022

Preprint

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Basic knowledge on the biology and epidemiology of equine strongylid species remains insufficient although it would contribute to the design of better parasite control strategies. Nemabiome is a convenient tool to quantify and to identify species in bulk samples that could overcome the hurdle that cyathostomin morphological identification represents. To date, this approach has relied on the internal transcribed spacer 2 (ITS-2) of the ribosomal RNA cistron and its predictive performance and associated biases both remain unaddressed. This study aimed to bridge this knowledge gap using cyathostomin mock communities and comparing performances of the ITS-2 and a cytochrome c oxidase subunit I (COI) barcode newly developed in this study. The effects of bioinformatic parameters were investigated to determine the best analytical pipelines. Subsequently, barcode predictive abilities were compared across various mock community compositions. The replicability of the approach and the amplification biases of each barcode were estimated. Results were also compared between various types of biological samples, i.e. eggs, infective larvae or adults. Overall, the proposed COI barcode was suboptimal relative to the ITS-2 rDNA region, because of PCR amplification biases, a reduced sensitivity and higher divergence from the expected community composition. Metabarcoding yielded consistent community composition across the three sample types, although infective larvae may remain the most tractable in the field. Additional strategies to improve the COI barcode performances are discussed. These results underscore the critical need of mock communities for metabarcoding purposes.

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“…DNA metabarcoding of faecal samples is emerging as a powerful method to mitigate issues arising from the difficulty of morphological worm identification, allowing the characterization of entire nemabiomes (nematode communities) by capturing DNA from eggs and larvae shed in faeces ( Avramenko et al, 2015 ). This method has been recently used to investigate horse nemabiomes ( Mitchell et al, 2019 ; Poissant et al, 2021 ), but has not yet been used in other equids.…”

Section: Introductionmentioning

confidence: 99%

The gastrointestinal nematodes of plains and Grevy's zebras: Phylogenetic relationships and host specificity

Tombak

Hansen

Kinsella³

et al. 2021

International Journal for Parasitology: Parasites and Wildlife

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Equids are chronically infected with parasitic strongyle nematodes. There is a rich literature on horse strongyles, but they are difficult to identify morphologically and genetic studies on strongyles infecting other equid species are few, hampering studies of host specificity. We sequenced expelled worms from two sympatric zebra species in central Kenya to expand the strongyle phylogeny and used DNA metabarcoding on faecal samples to genetically characterize zebra nemabiomes for the first time. We generated sequences for several species new to public genetic reference databases, all of which are typical strongyles in wild zebras (i.e., the three species of Cylindropharynx and Cyathostomum montgomeryi ), and identified their closest relatives. We also discovered an apparent fungus infecting a quarter of the expelled Crossocephalus viviparus worms, a hyperabundant nematode species in the family Atractidae, hinting at the possibility that zebra host-parasite dynamics may involve a zebra-fungus mutualism. The two zebra species had similar nemabiomes; we found a complete overlap in the list of nematode species they carry and very similar prevalence (i.e., proportion of hosts infected) for the different nematode species. Our study suggests limited host-specificity in zebra strongyles and high potential for transmission between the plains zebra and the endangered Grevy's zebra.

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A repeatable and quantitative DNA metabarcoding assay to characterize mixed strongyle infections in horses

Cited by 47 publications

References 44 publications

Characterizing parasitic nematode faunas in faeces and soil using DNA metabarcoding

Characterizing parasitic nematode faunas in faeces and soil using DNA metabarcoding

Evaluation of the nemabiome approach for the study of equine strongylid communities

The gastrointestinal nematodes of plains and Grevy's zebras: Phylogenetic relationships and host specificity

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