Estimation of Mycobacterium avium subsp. paratuberculosis Growth Parameters: Strain Characterization and Comparison of Methods

Abstract

ABSTRACTThe growth rate ofMycobacterium aviumsubsp.paratuberculosiswas assessed by different methods in 7H9 medium supplemented with OADC (oleic acid, albumin, dextrose, catalase), Tween 80, and mycobactin J. Generation times and maximum specific growth rates were determined by wet weight, turbidometric measurement, viable count, and quantitative PCR (ParaTB-Kuanti; F57 gene) for 8M. aviumsubsp.paratuberculosisstrains (K10, 2E, 316F, 81, 445, 764, 22G, and OVICAP 49). Strain-to-strain differences were observed in growth curves and calculated parameters. The quantification methods gave different results for each strain at specific time points. Generation times ranged from an average of 1.4 days for viable count and qPCR to approximately 10 days for wet weight and turbidometry. The wet-weight, turbidometry, and ParaTB-Kuanti qPCR methods correlated best with each other. Generally, viability has been assessed by viable count as a reference method; however, due toM. aviumsubsp.paratuberculosisclumping problems and the presence of noncultivableM. aviumsubsp.paratuberculosiscells, we conclude that qPCR of a single-copy gene may be used reliably for rapid estimation ofM. aviumsubsp.paratuberculosisbacterial numbers in a sample.