Identification and validation of reliable Aeromonas salmonicida subspecies salmonicida reference genes for differential gene expression analyses.

Aeromonas salmonicida subsp. salmonicida is a Gram-negative, facultative intracellular pathogen of a wide range of freshwater and marine fish species. A. salmonicida is the causative agent of furunculosis, an immunosuppressive disease that typically progresses to septicemia. Several aspects of A. salmonicida pathogenesis has already been described, but fundamental genetic aspects of the psychrophilic lifestyle of this bacterium remain unknown. Reverse transcription quantitative real-time polymerase chain reaction (qPCR) is a precise molecular technique used to detect very slight changes in gene expression. The appropriate choice of reference genes is essential for accurate normalization of qPCR gene expression data. Despite the available abundance of validated reference genes for mesophilic pathogens, a broad list of validated reference genes for A. salmonicida is not available. Here, we evaluated seven A. salmonicida reference genes under different culture conditions, including different growth phases, iron-limited and iron-supplemented conditions, and thermal stress. We determined that hfq maintained the most stable expression, followed by era, recA, rpoB, 16S, fabD, and gapA. The results of this study provided with an expanded list of reliable reference genes for A. salmonicida gene expression studies using qPCR.