Evaluation of a polymerase chain reaction-based assay for the detection of Aeromonas salmonicida ss salmonicida in Atlantic salmon Salmo salar

Abstract

A PCR-based assay was developed to detect Aerornonas salmonicida ss salmonicida (A.s.s.) in infected fish kidney and gills. Samples processed for PCR (polymerase chain reaction) were 100 ml kidney tissue suspensions and gill swabs. The primers and probes employed were derived from sequences of 16s rRNA as well as from plasmid DNA. In order to estimate in vitro sensitivity, various numbers of colony forming units (CFU) of A.s.s. strains were added to kidney and gill samples. A minimum of 20 and 200 CFU were demonstrated in 10 p1 PCR template by the 16s rDNA and the plasmid primers, respectively. The 20 and 200 CFU per 10 p1 PCR template correspond to 103 and 104 CFU in 100 m1 kidney tissue suspension. The in vitro specificity testing showed that DNA from A.s.s., A. hydrophila, A. salmonicida ss achromogenes, A. salmonicida ss masoucida, and atypical A. salmonicida were amplified using the 16s rDNA primers. Only A.s.s. and A. salrnonicida ss achromogenes DNA were amplified using the plasmid primers. Altogether 25 Atlantic salmon parr, experimentally challenged by cohabitation, were tested for the presence of A.s.s. by PCR and agar cultivation. The rates of recovery were 13/25 by PCR and 6/25 by agar cultivation. Kidney and gill samples from Atlantic salmon of about 2 kg, obtained at slaughter and considered covertly infected with A.s.s., were also tested. A.s.s. was detected neither by PCR nor by cultivation. Additionally, kidney samples from feral brood fish were tested. A.s.s. was more frequently, 24/72, detected by PCR than with agar cultivation, 6/72.