Sensitive and specific detection of viableMycobacterium aviumsubsp.paratuberculosisin raw milk by the peptide-mediated magnetic separation-phage assay

Abstract

The inclusivity (ability to detect all MAP strains), specificity (ability to detect only MAP) and detection sensitivity (ability to detect low numbers of MAP) of the optimized PMS-phage assay have been comprehensively demonstrated for the first time.

Are we closer to understanding why viable cells of Mycobacterium avium subsp. paratuberculosis are still being reported in pasteurised milk?

“…However, PCR assays while much more sensitive and faster than traditional culture methods are unable to differentiate between dead and living cells (Timms et al ), reducing their value when applied to heat‐treated milk and milk products. Because of these difficulties, phage amplification has been developed for the enumeration of viable MAP particularly in heated foods (Stanley et al ; Foddai and Grant, ; Gerrard et al, ).…”

“…However, PCR assays while much more sensitive and faster than traditional culture methods are unable to differentiate between dead and living cells (Timms et al 2011), reducing their value when applied to heat-treated milk and milk products. Because of these difficulties, phage amplification has been developed for the enumeration of viable MAP particularly in heated foods (Stanley et al 2007;Foddai and Grant, 2017;Gerrard et al, 2018). Stanley et al (2007) modified a commercial bacteriophage (phage)-based assay for M. tuberculosis (FASTPlaqueTB assay) using the broad-spectrum Mycobacterium phage D29 to detect viable cells of MAP in milk.…”

“…Stanley et al (2007) modified a commercial bacteriophage (phage)-based assay for M. tuberculosis (FASTPlaqueTB assay) using the broad-spectrum Mycobacterium phage D29 to detect viable cells of MAP in milk. Foddai and Grant (2017) have further developed this phage assay and reported the first relatively rapid, sensitive method for the enumeration of viable MAP using a peptide-mediated magnetic separation (PMS)-bacteriophage (phage) assay. This method was based on earlier studies designed to optimise a magnetic separation capture method using chemically synthesised MAP-specific peptides and the phage assay (Foddai et al 2009;Foddai et al 2010;Foddai et al 2011).…”

Viable Mycobacterium avium ssp. paratuberculosis isolated from calf milk replacer

“…As time passes and the novel optimized PMS-phage assay is applied to test various milk and dairy products, new information on the presence and numbers of viable MAP in these foods is emerging (Foddai and Grant, 2017). We previously reported the outcome of testing of powdered infant milk formula (PIF) by the PMS-phage assay (Grant et al, 2014).…”

“…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).…”