Optimization of a Phage Amplification Assay To Permit Accurate Enumeration of Viable Mycobacterium avium subsp. paratuberculosis Cells

Abstract

A commercially available phage amplification assay, FASTPlaqueTB (Biotec Laboratories, Ipswich, United Kingdom), when used according to the manufacturer's instructions, does not permit accurate enumeration of Mycobacterium avium subsp. paratuberculosis. The aim of this study was to optimize the phage amplification assay conditions to permit accurate quantification of viable M. avium subsp. paratuberculosis cells. The burst time for M. avium subsp. paratuberculosis was initially determined to inform decisions about optimal incubation time before plating, and then other test parameters were altered to evaluate how the correlation between plaque and colony counts was affected. The D29 mycobacteriophage replicates more slowly in M. avium subsp. paratuberculosis than in Mycobacterium smegmatis (used to optimize the commercial test originally), and the mean burst time for four M. avium subsp. paratuberulosis strains was 210 ؎ 36.8 min at 37°C compared to 63 ؎ 17.5 min for M. smegmatis mc 2 155. To achieve 100% correlation between plaque and colony counts, the optimized phage assay includes the following: (i) resuspension of the samples to be tested in Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase and 2 mM calcium chloride, followed by overnight incubation at 37°C before performance of the phage assay; (ii) a 2-h incubation of the sample with D29 mycobacteriophage before viricide treatment; and (iii) a further 90-min incubation after viricide treatment and neutralization up to the burst time (total incubation time, 210 min) before plating with M. smegmatis mc 2 155 in 7H9 agar. The optimized phage amplification assay was able to detect 1 to 10 CFU/ml of M. avium subsp. paratuberculosis in spiked milk or broth within 48 h, as demonstrated by the results of several blind trials.

Maximizing Capture Efficiency and Specificity of Magnetic Separation for Mycobacterium avium subsp. paratuberculosis Cells

“…paratuberculosis organisms in food or veterinary samples within 48 h using a commercially available phage amplification assay (FASTPlaqueTB assay; Biotec Laboratories Limited, Ipswich, United Kingdom), rather than waiting weeks for conventional culture results, is an exciting recent development (7,8,26). However, the mycobacteriophage used in the phage amplification assay has a broader mycobacterial host range than M. avium subsp.…”

Application of a Peptide-Mediated Magnetic Separation-Phage Assay for Detection of Viable Mycobacterium avium subsp. paratuberculosis to Bovine Bulk Tank Milk and Feces Samples

“…Recently, we optimized a peptide-mediated magnetic separation-phage (PMS-phage) assay to rapidly detect viable M. avium subsp. paratuberculosis (4,5). PMS selectively captures and concentrates M. avium subsp.…”

Viable Mycobacterium avium ssp. paratuberculosis isolated from calf milk replacer

“…The phage assay was carried out as described by Foddai et al (2009). Briefly, 100 µL of D29 mycobacteriophage suspension (10 9 pfu/mL) was added to each bead sample, before incubation for 2 h at 37°C.…”

“…However, detection methods for viable MAP in milk and dairy products have improved considerably over recent years with the advent of immunomagnetic separation (Grant et al, 1998;O'Brien et al, 2016) and subsequently peptide-mediated magnetic separation (PMS; Stratmann et al, 2002Stratmann et al, , 2006Foddai et al, 2010;O'Brien et al, 2016), which permit selective capture, separation, and concentration of whole MAP cells from a sample before culture or PCR, and novel mycobacteriophage-based methods of MAP detection (Stanley et al, 2007;Foddai et al, 2010;Swift et al, 2013;Botsaris et al, 2016), which require the MAP cells to be viable to obtain a positive result (plaques in a lawn of fast-growing Mycobacterium smegmatis). In particular, a method combining PMS and a phage amplification assay to detect MAP (PMS-phage assay), developed and optimized by Foddai et al (2009Foddai et al ( , 2011, and used in combination with an optimized milk sample preparation protocol (Foddai and Grant, 2015), is proving to be a very sensitive method of detecting viable MAP in cow milk. The optimized PMS-phage assay was recently reported to have a limit of detection 50% of ~1 MAP cell per 50 mL of milk, making it a more sensitive detection method than existing quantitative PCR for MAP and conventional culture methods (Foddai and Grant, 2017).…”