A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculos
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METHODS AND PROTOCOLS
A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows’ milk Antonio C. G. Foddai 1 & Irene R. Grant 1 Received: 29 July 2020 / Revised: 10 September 2020 / Accepted: 13 September 2020 # The Author(s) 2020
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-qPCRpositive, 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. Keywords Milk, Mycobacterium avium subsp. paratuberculosis (MAP), Phagomagnetic separation, Quantitative PCR (qPCR), Viability assay
Introduction Spread of Johne’s disease (JD) is becoming a big economic problem for dairy farmers worldwide (European Food Safety Authority 2017). JD (also known as Paratuberculosis) is a chronic intestinal infection caused by Mycobacterium avium subsp. paratuberculosis (MAP) which leads to chronic * Irene R. Grant [email protected] 1
Institute for Global Food Security, School of Biological Sciences, Queen’s University Belfast, Belfast, Northern Ireland BT9 5DL, UK
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. Animals generally become infected with MAP at a young age but can remain symptomless for several years. During this, time MA
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