Estimation of Mycobacterium avium subsp. paratuberculosis growth parameters: strain characterization and comparison of methods

Abstract

The growth rate of Mycobacterium avium subsp. paratuberculosis was 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 8 M. avium subsp. paratuberculosis strains (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 to M. avium subsp. paratuberculosis clumping problems and the presence of noncultivable M. avium subsp. paratuberculosis cells, we conclude that qPCR of a single-copy gene may be used reliably for rapid estimation of M. avium subsp. paratuberculosis bacterial numbers in a sample.

Fig. 1.

Growth curves obtained by quantification methods, wet weight (WW) (A), turbidometry (B), viable count (C), and qPCR (D), for vaccine and reference strains (■, 316F; ●, 2E; ▵, K10), bovine field isolates (○, 81; ♦, 445; □, 764), and ovine field isolates (▴, 22G; ♢, OC 49). McFar units, McFarland units; d, days. The error bars indicate standard deviations.

Fig. 2.

Time at which the maximum specific growth rate was achieved for each strain and quantification method. Black bars, wet weight; gray bars, turbidometry; hatched bars, viable count; open bars, qPCR.

Fig. 3.

Correlation between quantification methods for all strains (■, 316F; ●, 2E; ▵, K10; ○, 81; ♦, 445; □, 764; ▴, 22G; ♢, OC 49). The trend lines represent least squares estimation. All values were divided by the maximum value for each strain and quantification method.

Fig. 4.

(A) Growth curves monitored by DNA content for vaccine and reference strains (■, 316F; ●, 2E; ▵, K10), bovine field isolates (○, 81; ♦, 445; □, 764), and ovine field isolates (▴, 22G; ♢, OC 49). The error bars indicate standard deviations. (B) Correlation between qPCR (ParaTB-Kuanti) and DNA content. The trend line represents the least squares estimation. All values were divided by the maximum value for each strain and quantification method parameter.