Rapid quantitative detection of Aeromonas hydrophila strains associated with disease outbreaks in catfish aquaculture

A new strain of Aeromonas hydrophila has been implicated in significant losses in farm-raised catfish. Outbreaks attributable to this new strain began in Alabama in the summer of 2009 and have spread to Arkansas and Mississippi in subsequent years. These outbreaks mostly afflicted market-sized fish and resulted in considerable losses in short periods of time. The present research was designed to develop an expeditious diagnostic procedure to detect the new strains of A. hydrophila due to the rapid onset and biosecurity concerns associated with this new disease. A discriminatory quantitative polymerase chain reaction assay was developed using gene sequences unique to the virulent strains identified in a related comparative genomic study. Using this assay, suspect colonies on a culture plate can be positively identified as the new strain within 2 hr. The assay is repeatable and reproducible with a linear dynamic range covering 8 orders of magnitude and a sensitivity of approximately 7 copies of target DNA in a 15-µl reaction. In addition, the assay is able to detect and quantify the virulent strain from catfish tissues (0.025 g), pond water (40 ml), and sediments (0.25 g) with a sensitivity limit of approximately 100 bacteria in a sample. This assay provides rapid discrimination between the new virulent strain and more common A. hydrophila and is useful for epidemiological studies involving the detection and quantification of the virulent strain in environmental samples and fish tissues.