Which larvae are they? Use of single larva for the molecular confirmation of Cooperia pectinata and Cooperia punctata in Australian cattle

A 4 year observation of gastrointestinal nematode egg counts, nemabiomes and the benzimidazole resistance genotypes of Teladorsagia circumcincta on a Scottish sheep farm

“…Similarly, whilst Avramenko et al (2019) showed very high correlation between deep-amplicon sequencing and pyrosequencing of the β-tubulin locus and an allele detection limit of 0.1%, Sargison et al (2019) showed imperfect agreement between the expected and observed outcomes of deep-amplicon sequencing of mock pools of laboratory T. circumcincta isolates. The quantitative use of genetic speciation data derived from coprocultures is therefore not perfect, and Francis et al (2020) utilised a non-parametric approach to compare multiplexedtandem-PCR speciation against morphological identification of cattle GINs. The descriptive results and comparisons between years and ages in this study are supported by the additional nonparametric analyses; however there is a requirement for further studies that quantify the uncertainty around point estimates to support more powerful, parametric use.…”

Refugia or reservoir? Feral goats and their role in the maintenance and circulation of benzimidazole-resistant gastrointestinal nematodes on shared pastures

“…Stage-1 quantitative PCRs (qPCRs) for both the ITS-2 rDNA and isotype-1 β -tubulin nemabiome assays were performed on all gDNA lysates in-house (Francis et al ., 2020 ). Illumina adapter primers were previously described and validated (Avramenko et al ., 2015 ; Avramenko et al ., 2019 ).…”

“…Culture jars were humidified with ddH 2 O twice during incubation to ensure the feces retained sufficient moisture for larval development. Third-stage infective larvae (L3) were harvested as described in Francis et al (2020) and individual aliquots of 1000 L3 were stored overnight at 4°C. L3 samples were centrifuged at 1000 g for 2 min and total genomic DNA was extracted from the resulting pellets using the Monarch Genomic DNA Purification Kit (New England Biolabs, Australia).…”

“…ITS-2 rDNA species assignments and phylogenetic analysis ITS-2 ASVs were assigned to the species level using an in-house bespoke database of local trichostrongylid nematode ITS-2 rDNA reference sequences curated from previously published datasets derived from the GINs of livestock in New South Wales, Australia (Francis et al, 2020;Francis and Šlapeta, 2022) (LabArchives: https://dx.doi.org/10.25833/h6a4-be50). The database was imported into DADA2 using the 'assignTaxonomy' function, which assigns ASVs to the nearest match using the Bayesian classifier method, with a bootstrapping approach to assess the confidence assignment at each taxonomic level (Wang et al, 2007).…”