COMPARISON OF CULTURE PLATE METHOD AND PCR PROCEDURE TO DETECT A.salmonicida IN A FISH FARM ENVIRONMENT

Biochemical characterization of A.salmonicida was proved to show great homogenicity among strains of different origin and age. Plate culture and biochemical testing were proved to give reliable diagnostic but time duration for results oscillated between 96 hours and 10 days. Traditional plate culture techniques were compared with the novel molecular PCR procedure in the detection of the pathogenic bacteria Aeromonas salmonicida. Polymerase chain reaction (PCR) amplification of a 421 bp sequence from the surface array protein gene of A.salmonicida provided a specific and sensitive method for detection and identification of this important fish pathogen. The sensitivity of PCR detection of A.salmonicida in pure culture was found to be 30 cfu with a prior DNA extraction, and 120 cfu with only lysing the cells. For mix cultures sensitivity was found to decrease. by one order of magnitude. Nested PCR was found to increased PCR sensitivity by 10 fold in the pure culture, and by over 100 fold in mixed cultures. The PCR assay was not found to be either sensitive nor reliable on fish tissue samples and negative results were obtained for sediment samples. Inefficient cell lysis, DNA extraction and purification, and the presence of DNA polymerase inhibitors accounted for such poor results. "Nested PCR" on environmental samples gave positive results, however reliability was not confirmed due to the absence of a sensitivity experiment with positive controls. PCR was proved to be faster, more sensitive and specific than traditional plate culture assays, and therefore a better option for a routinary diagnostic technique to detect the presence of A.salmonicida. However alternative sample handling prior PCR is required to detect the pathogen reliably and in low numbers in fish tissue and environmental samples collected from a fish farm.Universidad de Santiago de Compostela Microbiology Departmen