The use of nemabiome metabarcoding to explore gastro-intestinal nematode species diversity and anthelmintic treatment effectiveness in beef calves

Abstract

Next-generation deep amplicon sequencing, or metabarcoding, has revolutionized the study of microbial communities in humans, animals and the environment. However, such approaches have yet to be applied to parasitic helminth communities. We recently described the first example of such a method - nemabiome sequencing - based on deep-amplicon sequencing of internal transcribed spacer 2 (ITS-2) rDNA, and validated its ability to quantitatively assess the species composition of cattle gastro-intestinal nematode (GIN) communities. Here, we present the first application of this approach to explore GIN species diversity and the impact of anthelmintic drug treatments. First, we investigated GIN species diversity in cow-calf beef cattle herds in several different regions, using coproculture derived L3s. A screen of 50 Canadian beef herds revealed parasite species diversity to be low overall. The majority of parasite communities were comprised of just two species; Ostertagia ostertagi and Cooperia oncophora. Cooperia punctata was present at much lower levels overall, but nevertheless comprised a substantive part of the parasite community of several herds in eastern Canada. In contrast, nemabiome sequencing revealed higher GIN species diversity in beef calves sampled from central/south-eastern USA and Sao Paulo State, Brazil. In these regions C. punctata predominated in most herds with Haemonchus placei predominating in a few cases. Ostertagia ostertagi and C. oncophora were relatively minor species in these regions in contrast to the Canadian herds. We also examined the impact of routine macrocyclic lactone pour-on treatments on GIN communities in the Canadian beef herds. Low treatment effectiveness was observed in many cases, and nemabiome sequencing revealed an overall increase in the proportion of Cooperia spp. relative to O. ostertagi post-treatment. This work demonstrates the power of nemabiome metabarcoding to provide a detailed picture of GIN parasite community structure in large sample sets and illustrates its potential use in research, diagnostics and surveillance.

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

“…This method is capable of discriminating between different nematode species based upon the sequence identity of the ITS-2 region of the rDNA. The samples were prepared and sequenced as described in Avramenko et al 2015 (Avramenko et al, 2015), and analysed with the bioinformatic pipeline described in Avramenko et al 2017 (Avramenko et al, 2017). Several A. caninum and A. braziliense ITS-2 sequences were added to the analysis database for the purposes of this analysis (GenBank accession: DQ438050, DQ438051, DQ438052, DQ438053, DQ438054, DQ438060, DQ438061, DQ438062, DQ438065, DQ438066, DQ438067, AB751614, AB751615, AB751616, DQ438072, DQ438073, DQ438074, DQ438075, DQ438076, DQ438077, DQ438078, DQ438079).…”

Why the COI barcode should be the community DNA metabarcode for the metazoa

“…It is largely for reasons of presumed taxonomic bias for PCR amplification of the COI‐bcr that many studies have abandoned this locus, in favour of primers matching highly conserved binding sites with a presumed more even coverage of all taxa present. The most widely used alternatives are the nuclear ribosomal genes coding for the small subunit (SSU or 18S rRNA) (Capra et al., ; Creer et al., ), the large subunit (LSU or 28S rRNA) (Hirai, Kuriyama, Ichikawa, Hidaka, & Tsuda, ), the internal transcribed spacer 2 (ITS2) (Anslan & Tedersoo, ; Avramenko et al., ) and the mitochondrial small [ rrnS or 12S rRNA] (Machida, Kweskin, & Knowlton, ) and large subunit rRNA [ rrnL or 16S rRNA] (Elbrecht et al., ; Saitoh et al., ). The lack of consensus over the choice of metabarcode markers, even within the same target community, carries the risk of poor standardization and low comparability among studies, which ultimately hampers the development of an efficient, universal system for biodiversity discovery and monitoring using cMBC.…”

A database for ITS2 sequences from nematodes

“…Consequently, deep amplicon sequencing approaches have a potentially powerful role for the investigation nematode communities similar to the use of bacterial 16S rDNA amplicon sequencing in microbiome studies. For example, the ITS2 rDNA locus has recently been used for "nemabiome" sequencing of parasitic nematode communities inhabiting the gastrointestinal tract of cattle (Avramenko et al, 2017). In that case, reliable species identification was achieved using a small bespoke, curated ITS2 rDNA database of the major relevant cattle gastrointestinal nematode species.…”