PCR-based assays for the fish pathogen Aeromonas salmonicida. II. Further evaluation and validation of three PCR primer sets with infected fish

Abstract

Two Aeromonas salmonicida-specific polymerase chain reaction (PCR) tests and 1 A. salmonicida subsp. salmonicida-specific PCR test were used to screen salmonid populations that were either overtly or covertly infected with A. salmonicida subsp. salmonicida. It was demonstrated that these PCR assays could be used to replace the biochemical testing currently employed to confirm the identity of A. salmonicida isolates cultured from infected fish. The AP and PAAS PCR assays were also capable of direct detection of A. salmonicida in overtly infected fish, with mucus, gill and kidney samples most likely to yield a positive result. Culture was a more reliable method for the direct detection of A. salmonicida in covertly infected salmonids than was the direct PCR testing of tissue samples, with the AP and PAAS PCRs having a lower detection limit (LDL) of approximately 4 × 10 5 colony-forming units (CFU) g -1 sample.

Molecular Detection and Characterization of Furunculosis and Other Aeromonas Fish Infections

“…The latter requires an intraperiotoneal injection of glucocorticoids (which generates immunosuppression) and then exposure of the fish to a heat shock (raising the temperature of the water to 18ºC for 14 days). This favours bacterial proliferation and the appearance of clinical signs, and will also increase the recovery rate of the bacteria (Austin & Austin, 2007;Bernoth et al, 1997;Byers et al, 2002b;Hiney & Olivier, 1999;Noga, 2010).…”

“…Skin and gills can also be cultured, but A. salmonicida may be uncovered by the presence of other dominant bacteria (Noga, 2010). False negatives in carrier fish are common since the bacteria are present in low concentrations (Byers et al, 2002b;Gustafson et al, 1992;Noga, 2010). More asymptomatic www.intechopen.com carriers can be detected by including either a pre-enrichment step before the PCR analysis, as commented before (Byers et al, 2002b;Gustafson et al, 1992) or by inducing stress using the approach named 'stress induced furunculosis' (SIF).…”

“…False negatives in carrier fish are common since the bacteria are present in low concentrations (Byers et al, 2002b;Gustafson et al, 1992;Noga, 2010). More asymptomatic www.intechopen.com carriers can be detected by including either a pre-enrichment step before the PCR analysis, as commented before (Byers et al, 2002b;Gustafson et al, 1992) or by inducing stress using the approach named 'stress induced furunculosis' (SIF). The latter requires an intraperiotoneal injection of glucocorticoids (which generates immunosuppression) and then exposure of the fish to a heat shock (raising the temperature of the water to 18ºC for 14 days).…”

“…Between 4 and 10 diseased fish are recommended to be sampled for detecting the pathogen, and between 10 and 60 fish in a population where there is a low mortality rate or the fish are apparently healthy (Noga, 2010). Detecting the pathogen in mucus, blood or faeces is also recommended because these samples do not require the fish to be sacrificed (Beaz-Hidalgo et al, 2008;Byers et al, 2002b;Gustafson et al, 1992;Kulkarni et al, 2009). When searching for asymptomatic carriers, the kidney and intestine are the recommended organs to sample.…”

“…Recognizing a bacterial fish pathogen requires their analysis in several organs (mainly kidney, but also spleen, skin, and ovaric fluid in breeding females) of a significant number of living fish (Byers et al, 2002b;Noga, 2010). Between 4 and 10 diseased fish are recommended to be sampled for detecting the pathogen, and between 10 and 60 fish in a population where there is a low mortality rate or the fish are apparently healthy (Noga, 2010).…”