Abstract
Bacteriophage-based methods for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis (MAP) in veterinary specimens are a recent addition to the Johne’s disease diagnostic toolbox. Here, we report the use of D29 mycobacteriophage-coated tosylactivated paramagnetic beads to capture and concentrate MAP cells from samples (termed phagomagnetic separation, PhMS) and then naturally lyse viable MAP cells (from the inside out) to provide DNA for IS900 qPCR purposes. Transmission electron microscopy confirmed that D29 phages had bound to beads in the correct orientation and that the phage-coated beads captured MAP cells from a suspension. During test optimization, conventional IS900 PCR results were used to subjectively assess the effect of different phage:bead coating ratios, differing amounts of coated beads during PhMS, optimal incubation time post-PhMS to obtain maximal MAP DNA, and the potential benefit of a brief heat shock (55 °C/1 min) prior to IS900 TaqMan qPCR. The limit of detection 50% (LOD50%) of the optimised PhMS-qPCR assay was 10.00 MAP cells/50 ml milk (95% CI 1.20–82.83). Finally, in order to demonstrate the new assay’s ability to detect viable MAP in naturally contaminated milk, bulk tank milk samples from 100 dairy farms were tested. Forty-nine (49%) of these tested PhMS-qPCR-positive, with viable MAP numbers detected ranging from 3–126 MAP/50 ml. The novel PhMS-qPCR assay is a sensitive, specific and easy-to-apply phage-based assay for viable MAP, with potential application for milk surveillance or diagnosis of Johne’s disease.Key points• Phage-coated magnetic beads could capture, concentrate and lyse MAP cells from milk.• PhMS-qPCR assay proved to be a rapid, sensitive and specific test for viable MAP.• A potential application of PhMS-qPCR assay for milk surveillance was demonstrated.
Highlights
Spread of Johne’s disease (JD) is becoming a big economic problem for dairy farmers worldwide (European Food Safety Authority 2017)
The objectives of this study were to (1) successfully coat D29 phages onto paramagnetic beads and use these phage-coated beads for phagomagnetic separation (PhMS) of Mycobacterium avium subsp. paratuberculosis (MAP) cells from milk; (2) determine the best protocol for harvesting DNA released from viable MAP cells lysed by action of the D29 phages; and (3) combine the PhMS and DNA harvesting steps with quantitative IS900 PCR to produce a rapid, sensitive and specific PhMS-Quantitative PCR (qPCR) assay for viable MAP in milk
The original FASTPlaqueTB assay for Mycobacterium tuberculosis and the peptide-mediated magnetic separation (PMS)-phage assay for MAP are examples of phage amplification assays
Summary
Spread of Johne’s disease (JD) is becoming a big economic problem for dairy farmers worldwide (European Food Safety Authority 2017). Paratuberculosis (MAP) which leads to chronic diarrhea, weight loss and declining milk production. Control of JD is currently very difficult due to the lack of sensitive tests able to detect early stages of infection. Time MAP is being shed in faeces and milk, leading to disease transmission within herds. As bacterial shedding precedes an antibody response against MAP, existing humoral tests can only detect animals in advanced stages of JD (Beaver et al 2017; Van Schaik et al 2003). MAP can be cultured from faeces, milk and blood of animals (Bower et al 2010; Gilardoni et al 2012), but because of the long doubling time of MAP, the method is slow and takes weeks to yield results.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have