A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows’ milk

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.

MAP, Johne’s disease and the microbiome; current knowledge and future considerations

“…Followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 20 s [ 75 , 97 ] mbtA_F3 (Forward 5′–CTC CCG CAA CTC GGT CAC–3) MAP2179_R3 (Reverse 5′–CAC AGC CAG GTG TGA AAG–3′) mbtA 307 1 cycle of pre-incubation: 95 °C for 10 min. Followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 20 s [ 97 ] P90 (Forward 5′-GAA GGG TGT TCG GGG CCG TCG GCC TTA GG- 3′) P91 (Reverse 5′-GGC GTT GAG GTC GAT CGC CCA CGT GAC-3') IS900 394 4 min of initial denaturation at 95 °C, 37 cycles of 95 °C for 30 s, 59.5 °C for 30 s and 72 °C for 30 s, followed by a final elongation at 72 °C for 4 min [ 31 , 101 ] AV1 (Forward 5'-ATGTGGTTGCTGTGTTGGATGG-3') AV2 (Reverse 5'-CCGCCGCAATCAACTCCAG-3') IS900 N/A 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 58 °C for 1 min and 72 °C for 1 min [ 28 , 31 , 101 ] …”

“…The assay is coupled with plaque PCR testing for the presence of signature elements and allows to detection of viable cells [ 104 ]. The Actiphage® test developed by Swift et al [ 107 ], which uses the above principles [ 108 ], has been commercialised by PBD Biotech with a similar method recently developed by [ 101 ]. Phage based assays can also be coupled with peptide-mediated magnetic separation for MAP cell capture [ 109 , 110 ].…”

Bacteriophage-Based Methods for Detection of Viable Mycobacterium avium subsp. paratuberculosis and Their Potential for Diagnosis of Johne's Disease

“…Two rapid, 1-day phage- and qPCR-based tests for viable MAP have been reported most recently - the Actiphage® Rapid assay ( 15 ) and the Phagomagnetic separation (PhMS)-qPCR assay ( 29 ). The Actiphage® technology is subject of a patent [( 30 ), PCT/GB2014/052970], and the Actiphage® Rapid assay is commercially available (PBD Biotech Limited).…”

“…Consistently, more viable MAP positive results are being obtained with the phage-based assays compared to culture, whether performed with prior chemical decontamination or PMS ( 20 , 24 , 27 ). The rapid, 1-day phage- and qPCR-based assays are proving to be more sensitive than either the Phage-PCR or PMS-phage assays, which are plaque assay-based tests ( 15 , 29 , 34 ). Phage assay results are often indicating the presence of viable MAP bacteraemia or MAP shedding in milk and feces in animals that are serum- or milk-ELISA negative ( 14 , 15 , 20 , 34 ).…”

The Application of Bacteriophage Diagnostics for Bacterial Pathogens in the Agricultural Supply Chain: From Farm-to-Fork

“…35 Phage capture has been used to detect MAP in BMT by fusing D29 phage to tosylactivated paramagnetic beads combined with qPCR testing; the limit of detection is reported as 10 MAP cells/50 mL of milk. 36 Magnetic separation was also incorporated into the phage amplification assay to isolate MAP cells from peripheral blood mononuclear cells 25,37 (Table 1). Technology transfer and optimization of the protocol caused variability in testing procedures; the magnetic separation step was thought to limit the sensitivity of the assay in blood samples.…”