Enumeration of MAP

Estimation of the number of MAP organisms that are present in a clinical sample can be used to determine the level of risk posed by livestock through contamination of the en­vironment.

Direct microscopic cell counts measure the total number of cells while PCR-based meth­ods measure total DNA levels, which can be related back to the total number of MAP cells. Both of these methods tend to overestimate counts by including both live and dead cells. In contrast, culture-based methods provide an estimate of the number of viable cells. Direct colony counts on HEYM slopes have often been used. Turbidimetric estimates, most probable number (MPN) estimates or time to growth are applicable to liquid cultures. As MAP tends to clump in suspension, counts from suspensions can be underestimates and considerable effort is required to achieve thorough dispersion of cells.

Turbidimetric estimates are only suitable for high concentrations of cells - one absorbance unit at 600 nm corresponds to a concentration of about 10⁸ cells/ml (Shin et al., 2007). Most probable number methods are applicable over a wide range of cell numbers but require the cul­ture of replicates of a serial dilution through to an endpoint, which is the highest dilution before growth no longer occurs; thus they are very cost­ly in terms of media (Whittington et al., 2000b; Reddacliff et al., 2003a). Estimation of count by the time to growth in radiometric BACTEC 12B medium is based on the observation that genera­tion time is proportional to inoculum size; this is less susceptible than other methods to error through bacterial clumping, is highly sensitive, can be done with a single culture vial and is ap­plicable to a range of sample types and strains of MAP (Lambrecht et al., 1988; Reddacliff et al., 2003a). The same approach has been used in MGIT media (Shin et al., 2007; Abendano et al.,

2012). Time to detection in the Trek ESP sys­tem is also related to inoculum size (Kim et al., 2002). Colony counts are likely to substantial­ly underestimate the actual count relative to MPN counts or time to growth in liquid media (Reddacliff et al., 2003a), although there was close correlation between counts on HEYM, BACTEC 12B medium and MGIT medium in one study (Shin et al., 2007).

To study pathogenesis and provide an in­direct measure of vaccine efficacy, Pooley et al. (2016) developed a liquid culture-based method for enumeration of MAP in intracellular infec­tion assays; it required a 2-week incubation and had a PCR readout.

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