What counts? A review of in vitro methods for the enumeration of Mycobacterium avium subsp. paratuberculosis

Abstract

Enumeration of Mycobacterium avium subsp. paratuberculosis (MAP) from clinical samples or from inoculum suspensions for challenge studies is an essential pre-requisite for paratuberculosis research. However, MAP organisms are slow and challenging to grow in vitro, with a strong tendency to clump. Standard bacteriological methods are not well suited to enumerate MAP. Enumeration can be achieved by culture-based methods, direct microscopic counts, turbidimetry, pelleted weights and quantitative PCR. There is an unresolvable discrepancy between culture-based methods, enumerating cultivable MAP organisms, and other methods enumerating total MAP organisms irrespective of viability, hence these methods are not directly comparable. However, they are complementary and should be used in parallel when accuracy is required. We review currently available methods, their principles, limitations and equivalence between methods.

Keywords: Enumeration; Epidemiology; Experimental studies; Interpretation; Mycobacterium avium subsp. paratuberculosis; Paratuberculosis.