Development of a PCR protocol for the detection of Aeromonas salmonicida in fish by amplification of the fstA (ferric siderophore receptor) gene

Molecular Detection and Characterization of Furunculosis and Other Aeromonas Fish Infections

“…All of them, except the subspecies pectinolytica have been implicated in fish pathology (Austin & Austin, 2007;Goldschmidt-Clermont et al, 2009;Han et al, 2011;Noga, 2010;Wiklund & Dalsgaard, 1998). Being unable to assign strains implicated in fish disease to any of the subspecies masoucida, smithia, achromogenes or pectinolytica using biochemical characteristics, they have been termed "atypical A. salmonicida" to differentiate them from the "typical A. salmonicida", a term restricted to the subspecies salmonicida (Beaz-Hidalgo et al, 2008;Goodwin & Merry, 2009;Wiklund & Daalsgard 1998). "Typical A. salmonicida" strains are psychrophilic, non-motile and produce brown pigment.…”

“…This problem was reported by other authors previously (Høie et al, 1997 and references therein) and could maybe be avoided including a pre-enrichment step (Byers et al, 2002b;Gustafson et al, 1992) or evaluating the quality of the extracted DNA for the presence of inhibitors (Mooney et al, 1995). However, none of these approaches were tested by Beaz-Hidalgo et al (2008). In the search for possible asymptomatic carrier fish, the fstA protocol was further assayed in the mucus and blood of 31 wild salmon that showed no signs of furunculosis, obtaining 4 A. salmonicida PCR-positive samples of mucus and 6 of blood recovered from 6 (19%) of the 31 salmon assayed (Table 1).…”

“…Detection limits of experimentally infected mucus and blood samples were 2.5 x 10 2 and 1.5 x 10 5 CFU/ml respectively. Bacteria present in seeded blood was only detected at a high concentration (1.5 x 10 5 CFU/ml), which is probably due to interferences with heparin or other unknown blood components that compete with the bacterial DNA in the PCR amplification (Beaz-Hidalgo et al, 2008). This problem was reported by other authors previously (Høie et al, 1997 and references therein) and could maybe be avoided including a pre-enrichment step (Byers et al, 2002b;Gustafson et al, 1992) or evaluating the quality of the extracted DNA for the presence of inhibitors (Mooney et al, 1995).…”

“…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.…”

“…Two new PCR methods were described in the first decade of the 21st century (Beaz-Hidalgo et al, 2008;Nilsson et al, 2006). The one of Nilsson and co-workers (2006) is based on the presence of an insertion element (ISE) in the tapA gene (encoding a pili subunit protein of the type IV pili) of A. salmonicida, which they named ISAsa4.…”