In order to introduce specificity for Mycobacterium avium subsp. paratuberculosis prior to a phage amplification assay, various magnetic-separation approaches, involving either antibodies or peptides, were evaluated in terms of the efficiency of capture (expressed as a percentage) of M. avium subsp. paratuberculosis cells and the percentage of nonspecific binding by other Mycobacterium spp. A 50:50 mixture of MyOne Tosylactivated Dynabeads coated with the chemically synthesized M. avium subsp. paratuberculosis-specific peptides biotinylated aMp3 and biotinylated aMptD (i.e., peptide-mediated magnetic separation [PMS]) proved to be the best magnetic-separation approach for achieving 85 to 100% capture of M. avium subsp. paratuberculosis and minimal (<1%) nonspecific recovery of other Mycobacterium spp. (particularly if beads were blocked with 1% skim milk before use) from broth samples containing 10 3 to 10 4 CFU/ml. When PMS was coupled with a recently optimized phage amplification assay and used to detect M. avium subsp. paratuberculosis in 50-ml volumes of spiked milk, the mean 50% limit of detection (LOD 50 ) was 14.4 PFU/50 ml of milk (equivalent to 0.3 PFU/ml). This PMS-phage assay represents a novel, rapid method for the detection and enumeration of viable M. avium subsp. paratuberculosis organisms in milk, and potentially other sample matrices, with results available within 48 h.The prospect of being able to detect viable Mycobacterium avium subsp. 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. paratuberculosis alone (23). Consequently, plaques obtained when naturally infected, rather than artificially spiked, samples are tested may not necessarily emanate from M. avium subsp. paratuberculosis alone if other Mycobacterium spp. are also present in the sample. Some additional selective step prior to phage infection, such as magnetic separation (12), is needed to introduce selectivity for M. avium subsp. paratuberculosis.Magnetic separation (MS) has become a routine method in food and veterinary microbiology laboratories and is commonly used in combination with culture or molecular methods for the detection and isolation of pathogenic bacteria such as Listeria monocytogenes (13, 31), Salmonella spp. (22, 25), and Escherichia coli O157:H7 in both the food (15) and veterinary (20) clinical sample testing context. Magnetic-separation methods selectively separate the target bacterium from other, nontarget microorganisms and inhibitory sample components while concentrating the target bacterial cells into a smaller volume. Collectively, these properties of magnetic separation enhance the analytical specificity and sensitivity of the ...
The ability to rapidly detect viable pathogens in food is important for public health and food safety reasons. Culture-based detection methods, the traditional means of demonstrating microbial viability, tend to be laborious, time consuming and slow to provide results. Several culture-independent methods to detect viable pathogens have been reported in recent years, including both nucleic acid-based (PCR combined with use of cell viability dyes or reverse-transcriptase PCR to detect messenger RNA) and phage-based (plaque assay or phage amplification and lysis plus PCR/qPCR, immunoassay or enzymatic assay to detect host DNA, progeny phages or intracellular components) methods. Some of these newer methods, particularly phage-based methods, show promise in terms of speed, sensitivity of detection and cost compared with culture for food testing. This review provides an overview of these new approaches and their food testing applications, and discusses their current limitations and future prospects in relation to detection of viable pathogens in food. Key points • Cultural methods may be 'gold standard' for assessing viability of pathogens, but they are too slow. • Nucleic acid-based methods offer speed of detection but not consistently proof of cell viability. • Phage-based methods appear to offer best alternative to culture for detecting viable pathogens.
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.
Naturally contaminated bovine bulk tank milk (n ؍ 44) and feces (n ؍ 39) were tested for the presence of viable Mycobacterium avium subsp. paratuberculosis by a novel peptide-mediated magnetic separation-phage (PMS-phage) assay. Counts of viable M. avium subsp. paratuberculosis cells ranging from 1 to 110 PFU/50 ml of milk and 6 to 41,111 PFU/g of feces were indicated by the PMS-phage assay.Culture is considered the definitive diagnostic test for Mycobacterium avium subsp. paratuberculosis in diagnostic specimens such as feces and bulk tank milk (BTM) (18). However, it is labor intensive and time consuming and involves chemical decontamination and several months of incubation. 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. paratuberculosis cells from a sample, effectively removing the vast majority of contaminating microorganisms, and the phage amplification assay enables rapid enumeration of viable M. avium subsp. paratuberculosis cells within 24 h. When employed together, PMS and the phage amplification assay are selective for low numbers of viable M. avium subsp. paratuberculosis cells in artificially spiked milk samples (5) without the need for chemical decontamination. The objective of this study was to assess the performance of this novel PMS-phage assay when applied to naturally contaminated bovine BTM and feces samples.Fresh BTM samples (n ϭ 25) from dairy farms in Northern Ireland (NI) on which one or more animals were seropositive for M. avium subsp. paratuberculosis and previously frozen (Ϫ70°C for several months) BTM (n ϭ 19) and feces samples (n ϭ 39) from dairy herds of known Johne's disease status connected with the University of Pennsylvania School of Veterinary Medicine were tested. A 50-ml aliquot of each BTM sample was centrifuged at 2,500 ϫ g for 15 min and the pellet was resuspended in 1 ml of Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase (OADC) prior to the optimized PMS-phage assay. One gram of feces was mixed thoroughly with 4 ml of sterile water, and the sample was subjected to low-speed centrifugation (300 ϫ g for 3 min). One milliliter of the resultant supernatant was processed through the optimized PMS-phage assay (5). PMS beads were resuspended in Middlebrook 7H9 broth containing a nystatinoxacillin-aztreonam (NOA) antimicrobial supplement (final concentrations per ml, 50 IU of nystatin, 2 g of oxacillin, and 30 g of aztreonam; Biotec Laboratories Limited, United Kingdom) to limit the growth of background microflora during the subsequent incubation steps. Plaques were counted and expressed as PFU/50 ml of BTM and PFU/g of feces. Plaque-IS900 PCR (15) was performed on PMS-phage assay-positive samples to confirm the presence of M. avium subsp. paratuberculosis DNA.BTM samples were also cultured. A 50-ml aliquot of each NI BTM sample was decontaminated with 0.75% hexadecylpyridinium chloride (HP...
26Hands can be a vector for transmitting pathogenic microorganisms to foodstuffs and 27 drinks, and to the mouths of susceptible hosts. Hand washing is the primary barrier The aim of this systematic review was to collate scientific information on the efficacy
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