Strongylus vulgaris

Harvey, Andrea M., Meggiolaro, Maira N., Hall, Evelyn, Watts, Ellyssia T., Ramp, Daniel & Šlapeta, Jan, 2019, Wild horse populations in south-east Australia have a high prevalence of Strongylus vulgaris and may act as a reservoir of infection for domestic horses, International Journal for Parasitology: Parasites and Wildlife 8, pp. 156-163 : 157-159

2.5. Strongylus vulgaris diagnostic qPCR

DNA was isolated from 134 faecal samples across the six populations using the MagAttract PowerMicrobiome DNA/RNA Kit (Cat No./ ID: 27600-4-KF, Qiagen, Australia) optimised for KingFisher º Duo. Each sample was homogenized for 40 s at 6.0 m/s on a FastPrep-24 benchtop homogeniser (MP Biomedicals, Australia). Samples were extracted in batches of 12 including an extraction blank per batch. DNA was eluted into 100 μL stored at −20 ̊C.

The diagnostic S. vulgaris qPCR was performed using described methodology ( Nielsen et al., 2008). The qPCR reaction included 2 μL of DNA template in a total volume of 20 μL using the SensiFAST™ Probe No-ROX Kit (Cat No.: BIO-86020, Bioline, Australia). The oligonucleotides used in a final concentration of 400 nM for primers and 100 nM for the probe per reaction were: Forward primer 5′ -GTA TAC ATT AAA TAG TGT CCC CCA TTC TAG- 3’; Reverse primer 5′ -GCA AAT ATC ATT AGA TTT GAT TCT TCC G- 3’; Probe 5′-FAM-TGG ATT TAT TCT CAC TAC TTA ATT GTT TCG CGA C-BHQ3-3’. Primers and probe were synthesized by Macrogen (Seoul, Korea). The qPCR conditions included 95 ̊C for 3 min, followed by 40 cycles of 95 ̊C for 10 s and 60 ̊C for 30 s. The qPCR assay was run on CFX96 TouchTM Real-Time PCR Detection System (BioRad, Australia), with samples run in duplicate. Real-time PCR results were analysed using BioRad CFX Manager 3.1 (BioRad, Australia). Positive results were determined if one or more repeats yielded Ct values <40.00 and negative results were

spp. faecal egg counts in each population.

SE Standard Error.

EPG Eggs per gram.

Low density = estimated <1 horse/km2, medium density = estimated 1–2 horses/km2, high density = estimated> 2 horses/km2 ( Cairns and Robertson, 2015;

Watts, 2017).

present or absent. Anoplocephala spp . and S. vulgaris PCR results were analysed separately using a General Linear Mixed Model with a binomial distribution. Fixed effects were Location, Density and Habitat, with the random effect of Sample.

determined if both repeats did not cross the threshold (Ct ≥ 40). Each qPCR run included positive control from DNA extracted from known adult S. vulgaris (courtesy of Ian Beveridge, University of Melbourne), negative control (water), plus the DNA extraction blank control.