Abstract
Dichelobacter nodosus is the primary etiological agent of footrot in sheep and has a variety of virulence factors. Of these, AprV2, an extracellular protease, has been shown to be capable of causing severe or “virulent” disease symptoms under the right conditions. Due to this, a loop-mediated isothermal amplification (LAMP) assay for the detection of aprV2-positive D. nodosus (VDN LAMP) was developed and evaluated for field use. A sample of 19 sheep flocks (309 sheep) in Victoria, Australia, were tested to determine the optimum conditions for in-field VDN LAMP assay use and sampling, for detecting aprV2-positive D. nodosus infected sheep. VDN LAMP performance was compared to a validated rtPCR that detects aprV2 and the benign strain counterpart, aprB2, using biologically duplicate samples to determine sensitivity and specificity. Flocks were sampled either in winter-spring (moist) or early summer (dry) conditions and had a range of clinical expressions of the disease ovine footrot. Variables considered for optimizing field performance were: sample collection method, sample preparation, clinical expression of disease, and nature of the feet when sampled (moist vs. dry, clean vs. soiled). The test was found to perform best when sheep were sampled with moist, clean feet, using a dry swab with the sample prepared in alkaline polyethylene glycol, pH 13.0, as the collection buffer. A sensitivity of 89% and specificity of 97% was seen when used in-field under these conditions, when compared to aprV2 detection by rtPCR, with “very good” agreement to rtPCR results. This study shows the VDN LAMP test is easy to use in-field to identify the presence of aprV2-positive D. nodosus in sheep flocks.
Highlights
Footrot in sheep is a disease that causes considerable welfare and economic concerns
From the nine methods investigated for field processing of samples, a swab collected into 500 μL alkaline polyethylene glycol (PEG), pH 13.0, was the easiest sampling method to perform with the least amount of equipment
The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the in-field VDN loop-mediated isothermal amplification (LAMP) was calculated by direct comparison with the aprV2 Real-time polymerase chain reaction (rtPCR) Ct values as determined from the biologically duplicate swabs
Summary
Footrot in sheep is a disease that causes considerable welfare and economic concerns. Dichelobacter nodosus, a gram-negative bacterium, is the primary etiological agent and causes the hard hoof to separate or “underrun” from the soft underlying tissue of the sheep foot [1]. In Australia, clinical footrot is divided into two forms—benign and virulent. This is referring to the visual symptoms of D. nodosus infection and in Victoria is based on a modified Egerton scoring system of 1–5 [2]. Footrot is spread over the pasture, in Spring in Victoria (September– November), posing a biosecurity threat both on the farm between flocks and in the larger industry [4, 5]
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