Detection and Enumeration of Mycobacterium avium subsp paratuberculosis in Milk

Different detection methods for M. a. paratuberculosis have been evaluated. Enumeration of cells was performed by Direct Epifluorescence Filter Technique (DEFT) microscopy using acridine orange staining and solid phase cytometry (ChemScan; Chemunex, Maisons-Alfort, France) after viability labelling with a fluorogenic substrate for esterase. Similar results were obtained with DEFT and ChemScan (maximum difference with a factor 4.2 for dispersed cells after physical disruption of the clumps and with a factor 2.3 for clumps). The number of cells after spiking in raw milk was estimated by a BactoScan 8000 (Foss Electric, Hillerod, Denmark). This approach yielded a considerable underestimation with reference to ChemScan (underestimation factor 1.2 to 14.3) and was more variable because the intensity of the fluorescence for a portion of cells was below the setting threshold of the apparatus. The sensitivity of PCR detection for clumps after enzymatic and chemical destruction of raw milk was estimated as 4 - 8 cfu/ml and 40 - 800 cfu/ml, respectively, while the sensitivity of conventional culturing was estimated as 130 cfu/ml.