Validation of ITS-2 rDNA nemabiome sequencing for ovine gastrointestinal nematodes and its application to a large scale survey of UK sheep farms

Redman, Elizabeth; Queiroz, Camila; Bartley, David J.; Levy, Morris; Avramenko, Russell W.; Gilleard, John S.

doi:10.1016/j.vetpar.2019.108933

Cited by 60 publications

(73 citation statements)

References 21 publications

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“…Sequence data was analysed using a bioinformatics pipeline based on Mothur version 1.36.1 [23] as previously described [24,25]. In brief, raw paired-end reads were assembled into single contigs and then filtered to removed contigs < 200 bp or > 450 bp or those with ambiguities between the overlapping paired-end reads.…”

Section: Bioinformatics Analysismentioning

confidence: 99%

High levels of third-stage larvae (L3) overwinter survival for multiple cattle gastrointestinal nematode species on western Canadian pastures as revealed by ITS2 rDNA metabarcoding

et al. 2020

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Background The ability of infective larvae of cattle gastrointestinal nematode (GIN) species to overwinter on pastures in northerly climatic zones with very cold dry winters is poorly understood. This is an important knowledge gap with critical implications for parasite risk assessment and control. Methods Infective third-stage larvae (L3) were quantified in samples of fecal pats, together with adjacent grass and soil, before and after winter on three farms in southern, central and northern Alberta. Nemabiome ITS2 metabarcoding was then performed on the harvested L3 populations to determine the species composition. Finally, parasite-free tracer calves were used to investigate if the L3 surviving the winter could infect calves and develop to adult worms in spring. Results Farm level monitoring, using solar powered weather stations, revealed that ground temperatures were consistently higher, and less variable, than the air temperatures; minimum winter air and ground temperatures were − 32.5 °C and − 24.7 °C respectively. In spite of the extremely low minimum temperatures reached, L3 were recovered from fecal pats and grass before and after winter with only a 38% and 61% overall reduction over the winter, respectively. Nemabiome ITS2 metabarcoding assay revealed that the proportion of L3 surviving the winter was high for both Cooperia oncophora and Ostertagia ostertagi although survival of the former species was statistically significantly higher than the latter. Nematodirus helvetinaus and Trichostrongylus axei could be detected after winter whereas Haemonchus placei L3 could not overwinter at all. Adult C. oncophora, O. ostertagi and N. helvetianus could be recovered from tracer calves grazing after the winter. Conclusions The largest proportion of L3 were recovered from fecal pats suggesting this is important refuge for L3 survival. Results also show that L3 of several GIN parasite species can survive relatively efficiently on pastures even in the extreme winter conditions in western Canada. Tracer calf experiments confirmed that overwintered L3 of both C. oncophora and O. ostertagi were capable of establishing a patent infection in the following spring. These results have important implications for the epidemiology, risk of production impact and the design of effective control strategies. The work also illustrates the value of applying ITS2 nemabiome metabarcoding to environmental samples.

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Section: Bioinformatics Analysismentioning

confidence: 99%

High levels of third-stage larvae (L3) overwinter survival for multiple cattle gastrointestinal nematode species on western Canadian pastures as revealed by ITS2 rDNA metabarcoding

et al. 2020

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“…Still, it is unclear where to de ne the cut-off for discriminating between nematode species, as genetic isolation is generally used to de ne species boundaries rather than DNA differences [40]. Nevertheless, in agreement with previously studies on nemabiome in livestock and horses [16][17][18][19][20], a cut-off identity threshold of ≥ 99 seems reasonable. However, as shown in our study, the query cover also needs to be considered.…”

Section: Discussionmentioning

confidence: 64%

“…Thus, both the epidemiological consequences and clinical impact of these minor species are probably of less importance for Swedish conditions. With the exception of T. colubriformis, which was absent in our data set, we recognized all eight major species found in UK sheep based on data generated with a similar approach [19]. However, the relative abundance of the distinctive species differed.…”

Section: Discussionmentioning

confidence: 94%

“…Like for any microbe NGS allows for the characterization of complex nematode communities and it open up new possibilities to identify community components even at low relative abundances at an unprecedented depth with minimal cost and labor. Recently the term "nemabiome" was created to describe the community structure of nematodes in ruminant livestock [16], By using an Illumina based deep amplicon next generation pooled sequencing approach targeting ITS-2 rDNA locus of the nematode rRNA genes, a pipeline was developed which so far has been used to study the entire nemabiome in beef cattle [16], bison [17] and dairy cattle [18] in Canada and the US, as well as in UK sheep [19]. Similarly, the nemabiome of equines were recently studied by a slightly different methodology [20].…”

Section: Introductionmentioning

confidence: 99%

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Sheep Nemabiome Diversity and Its Response to Anthelmintic Treatment in Swedish Sheep Herds

Halvarsson¹,

Höglund

2020

Preprint

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Background: A novel way to study the species composition and diversity of nematode parasites in livestock is to perform deep sequencing on composite samples containing a mixture of different species. Herein we describe for the first-time the nematode community structure (nemabiome) inhabiting Swedish sheep and how these are/were affected by host age and recent anthelmintic treatments. Methods: A total of 158 larval cultures were collected (n=35 in 2007 and n=126 in 2014-2016) from groups of sheep on 61 commercial farms in the south-central part of country were most animals are grazed. Among the samples, 2 x 44 (56%) were paired collected from the same groups pre- and post-treatment with macrocyclic lactones, benzimidazoles or levamisole. The sequences were analyzed for their nemabiome using the PacBio platform followed by bioinformatic sequence analysis with SCATA. Species richness and diversity were calculated and analyzed in R.Results: Nematode ITS2 sequences were found in all samples except two, despite that the fecal egg counts were below the McMaster threshold in 20 samples. Sequencing yielded on average 1,011 sequences per sample. Totally 26 operational taxonomical units (OTU) among which 18 (69%) had ≥99 % identity to sequences in the NCBI database were recognized. The OTUs found represented nematode species among which 10 are commonly associated with sheep. Multiple species were identified in all pre anthelmintic treatment samples. No effects on nematode diversity were found in relation to host age. On the other hand, recent anthelmintic treatment lowered species richness, especially after use of IVM and ABZ. Interestingly, despite zero egg count after use of levamisole, these samples still contained nematode DNA and especially H. contortus. Conclusions: Our findings provide evidence for that nemabiome analysis combined with diversity index analysis provide a sensitive and objective methodology in the study of the efficacy of anthelmintic treatment.

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“…Recently the term "nemabiome" was created to describe the community structure of nematodes in ruminant livestock [16]. By using an Illumina based deep amplicon next generation pooled sequencing approach targeting the nematode ITS2 rRNA gene, a pipeline was developed which so far has been used to study the entire nemabiome in beef cattle [16], bison [17] and dairy cattle [18] in Canada and the US, as well as in UK sheep [19]. Similarly, the nemabiome of equines were recently studied by a slightly different methodology [20].…”

Section: Like For Any Microbe Ngs Allows For the Characterization Ofmentioning

confidence: 99%

Sheep nemabiome diversity and its response to anthelmintic treatment in Swedish sheep herds

Halvarsson¹,

Höglund

2020

Preprint

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BackgroundA novel way to study the species composition and diversity of nematode parasites in livestock is to perform deep sequencing on composite samples containing a mixture of different species. Herein we describe for the first-time the nematode community structure (nemabiome) inhabiting Swedish sheep and how these are/were affected by host age and recent anthelmintic treatments.MethodsA total of 158 fecal samples were collected (n=35 in 2007 and n=123 in 2013-2016) and cultured from groups of sheep on 61 commercial farms in the south-central part of country where most animals are grazed. Among the samples, 2 x 44 (56%) were paired collections from the same groups pre- and post-treatment with macrocyclic lactones, benzimidazoles or levamisole. The sequences were analyzed for their nemabiome using the PacBio platform followed by bioinformatic sequence analysis with SCATA. Species richness and diversity were calculated and analyzed in R.ResultsNematode ITS2 sequences were found in all larval culture samples except two, even though the fecal egg counts were below the McMaster threshold in 20 samples. Sequencing yielded, on average, 1,008 sequences per sample. In total 16 operational taxonomical units (OTU), all had ≥98 % identity to sequences in the NCBI database, were recognized. The OTUs found represented nematode species of which 10 are commonly associated with sheep. Multiple species were identified in all pre anthelmintic treatment larval culture samples. No effects on nematode diversity were found in relation to host age. On the other hand, recent anthelmintic treatment lowered species richness, especially after use of ivermectin and albendazol. Interestingly, despite zero egg counts after use of levamisole, these samples still contained nematode DNA and especially H. contortus.ConclusionsOur findings provide evidence that nemabiome analysis combined with diversity index analysis provide a sensitive and objective methodology in the study of the efficacy of anthelmintic treatment, as both high and low abundant species were detected.

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Validation of ITS-2 rDNA nemabiome sequencing for ovine gastrointestinal nematodes and its application to a large scale survey of UK sheep farms

Cited by 60 publications

References 21 publications

High levels of third-stage larvae (L3) overwinter survival for multiple cattle gastrointestinal nematode species on western Canadian pastures as revealed by ITS2 rDNA metabarcoding

High levels of third-stage larvae (L3) overwinter survival for multiple cattle gastrointestinal nematode species on western Canadian pastures as revealed by ITS2 rDNA metabarcoding

Sheep Nemabiome Diversity and Its Response to Anthelmintic Treatment in Swedish Sheep Herds

Sheep nemabiome diversity and its response to anthelmintic treatment in Swedish sheep herds

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