Growth dynamic curve and gene expression analysis for optimized culture medium of Mycoplasma hyopneumoniae CJ strain

Mycoplasma hyopneumoniae is the primary etiologic agent of swine enzootic pneumonia, and vaccines remain the cornerstone of disease control. Industrial production of the pathogen, however, is severely constrained by its intrinsically slow growth, fastidious nutrient demands, and the lack of robust real-time cell counts quantification. Conventional color change unit assays require 14 days of incubation, exhibit poor reproducibility, and preclude dynamic optimization of cultivation processes. In this study, the following aspects were addressed. First, two optimized Friis-based media, CL6 and GD-240, were formulated. Second, a dynamic monitoring platform integrating a logistic growth model with flow cytometry was developed to enable real-time tracking of mycoplasma growth. Five key growth parameters were defined, and kinetic profiling revealed that GD-240 increased maximum cell counts by 17.5% and prolonged the rapid growth phase by 2.7 h. Third, quantitative PCR was used to clarify the temporal expression patterns of core metabolic and virulence genes; for instance, deoB and ldh were found to be upregulated at 30 h and downregulated at 42 h. These practical methodological approaches were provided for the optimization of mycoplasma cultivation and dynamic physiological analysis.