Abstract
Information on the in vitro growth of pathogenic and non-pathogenic Mycoplasma hyopneumoniae (M. hyopneumoniae) strains is scarce and controversial. Despite its limitations, the colour changing units (CCU) assay is still considered the golden standard titration technique for M. hyopneumoniae culture. Thus, the aims of the present study were: (1) to describe the growth dynamics and kinetics of pathogenic and non-pathogenic M. hyopneumoniae strains, and (2) to monitor the strains’ daily growth by ATP luminometry, CCU, colony forming units (CFU), and DNA quantification by real time quantitative PCR (qPCR) and by fluorescent double-stranded DNA (dsDNA) staining, to evaluate them as putative titration methodologies. The growth of the non-pathogenic J (ATCC®25934™) type strain and the pathogenic 11 (ATCC®25095™) reference strain and 232 strain was modelled by the Gompertz model. Globally, all three-strain cultures showed the same growing phases as well as similar maximal titres within a particular technique, but for CFU. However, the J strain displayed the fastest growing. During the logarithmic phase of growing, CCU, ATP and M. hyopneumoniae copy titres were strongly and linearly associated, and correlation between techniques could be reliably established. In conclusion, real-time culture titration by means of ATP or molecular assays was useful to describe the in vitro growth of the tested strains. Knowledge about the in vitro growth behaviour of a specific strain in a specific medium may provide several advantages, including information about the time required to reach maximal titres by the culture. Noteworthy, the obtained results refers to the three strains used, so extrapolation to other M. hyopneumoniae strains or culture conditions should be made cautiously.
Highlights
In 1965, Maré and Switzer in the United States and Goodwin in the United Kingdom isolated Mycoplasma hyopneumoniae (M. hyopneumoniae) strains 11 and J, respectively [1]
A fungus-like contamination was detected in replicate A of M. hyopneumoniae J strain culture at D7
It is important to mention that ATP concentration levels between strain initial inocula after thawing were very similar (1222.1 ± 86.2 pmol ATP/mL), which verified that the growth dynamics was started from approximated equal conditions for each M. hyopneumoniae strain culture
Summary
In 1965, Maré and Switzer in the United States and Goodwin in the United Kingdom isolated Mycoplasma hyopneumoniae (M. hyopneumoniae) strains 11 and J, respectively [1]. Low-passaged M. hyopneumoniae strains may grow slowly and may yield lower number of cells in medium than higherpassaged strains, which, in turn, may not be virulent [3, 14, 15]. In connection with this matter, non-pathogenic strains’ protein expression profile has been shown to be more related to metabolism than to infectiousness [10, 11]. The putative relationship between pathogenicity and in vitro growing capacity of M. hyopneumoniae deserves further investigation
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