Rapid enumeration of viable Mycobacterium avium subsp paratuberculosis in milk

Mycobacterium avium subsp. paratuberculosis (Map) is suspected of playing a role in human Crohn's disease and of surviving industrial pasteurisation of milk. Although PCR and culture methods are able to detect Map in milk, no method for the rapid detection of viable whole cells in milk has been established thus far. We have developed a method based on solid phase cytometry (SPC) to enumerate Map in 50 ml of spiked pasteurised milk within a single working day. SPC is an innovative technique which allows the enumeration of fluorescently labelled bacteria as single cells on a membrane filter. The SPC procedure consists of a membrane filtration, the labelling of bacteria on the membrane filter with an Ar laser excitable fluorescent dye, the automated counting in 3 min by the ChemScan apparatus (Chemunex, Ivry-sur-Scine, France) and a visual confirmation by means of an epifluorescence microscope. An extensive sample clean-up was developed in order to render the milk filterable and to eliminate the predominant background flora. This pretreatment consisted of a chemical milk destruction, several centrifugation steps, decontamination with 0.75 % cetyl pyridinium chloride, an enzymatic treatment, a prefiltration step and a purification by means of hydrophobic C-8 polymeric beads. In the actual SPC procedure, the Map retained on the membrane filter were fluorescently labelled using the non-specific viability substrate ChemChrome V6 which stains all living microorganisms. Therefore, the selectivity of the method depends on sample pretreatment only, i.e. milk destruction, decontamination and isolation on C-8 beads. For spiked pasteurised milk inoculated with 10(3)-10(5) mycobacterial cells recoveries between 10-50 % were obtained. On-going research focuses on a selective labelling procedure and the detection of Map in naturally contaminated milk.