241
and recorded 87 % positives, with 100 genome equivalents/fi sh, using a specifi c
DNA probe for Aer. salmonicida . Høie et al. ( 1997 ) designed primers and probes
from 16S rRNA and plasmid DNA; the former of which amplifi ed Aer. hydrophila,
Aer. salmonicida subsp. achromogenes, Aer. salmonicida subsp. masoucida and
atypical isolates, whereas the latter detected only Aer. salmonicida subsp. achromogenes and Aer. salmonicida subsp. salmonicida. Based on an examination of 100 ml
volumes of kidney suspension and gill swabs, the PCR detected 20 and 200 colony
forming units in 10 μl of PCR template by 16S rRNA and plasmid primers, respectively. The numbers corresponded to 10
3 and 10
4 colony forming units in 100 ml of
kidney suspension, respectively (Høie et al. 1997 ). A conclusion was reached that
the PCR detected Aer. salmonicida more often than culturing. Terminal-RFLP permitted the detection of ~30 CFU/mg of artifi cially inoculated kidney tissue (Nilsson
and Strom 2002 ). A multiplex PCR was developed for the simultaneous detection
Aer. salmonicida, Pis. salmonis, Str. phocae and V. anguillarum . The detection limit
using purifi ed total bacterial DNA was 5 pg/ μl (=5.33 × 10
4 CFU/ml). The limits of
detection using spiked tissues, i.e. kidney, liver, muscle or spleen, were
3.8 ± 0.78 × 10
3 CFU/mg (Tapia-Cammas et al. 2011 ). A possible drawback for use
of DNA probes, however, has been proposed by Hennigan et al. ( 1989 ). Using four
DNA probes in combination with seven restriction enzymes and seven strains of
Aer. salmonicida, their data suggested that the DNA sequences of the species is very
strongly conserved. They emphasised that the use of DNA probe technology to
identify different strains of Aer. salmonicida may be limited. Using reverse
transcription- multiplex PCR with primers, SV1/SV2 and SF1/SF2, which were specifi c to vapA and fstB genes, respectively, Rattanachaikunsopon and Phumkhachorn
( 2012 ) detected 10 CFU in pure culture and 30 CFU in tissue. Moreover, the method
distinguished viable from non-viable cells, and typical from atypical representatives
of Aer. salmonicida. Keeling et al. ( 2013 ) developed and validated a highly specifi c
and reproducible real-time PCR, which detected the surface array protein, vapA,
with reported sensitivity of 5 fg of DNA, 2.2 × 10
4 CFU/g of kidney tissue without
enrichment and 40 CFU/g with enrichment.
In a comparison of the sensitivity of culturing with DNA probes, the former
detected Aer. salmonicida from the kidney of only dead or moribund farmed Atlantic
salmon smolts in Ireland whereas probe technology allied to a PCR assay was capable of recognising the pathogen in water, faeces and effl uent (O’Brien et al. 1994 ).
The benefi t of species-specifi c primers and a nested PCR was demonstrated over
universal eubacterial primers when the detection limit improved from 1.4 × 10
4 CFU/
reaction to <14 CFU/sample (Taylor and Winton 2002 ). The importance of the
primer set was further highlighted in a comparative study by Byers et al. ( 2002a , b ).
Denaturing Gradient Gel Electrophoresis (DGGE) A method has been proposed and was evaluated in wild spawning coho salmon ( Oncorhynchus kisutch )
and cultured lake trout ( Salvelinus namaycush ) for the nonlethal detection of the
pathogen from mucus involved DGGE of 16S rDNA, which led to a reproducible
4-band pattern. This was distinctive from other aeromonads, and Aer. salmonicida.
The technique recognised 36 out of 52 coho salmon that were positive for Aer. salAeromonas salmonicida
and recorded 87 % positives, with 100 genome equivalents/fi sh, using a specifi c
DNA probe for Aer. salmonicida . Høie et al. ( 1997 ) designed primers and probes
from 16S rRNA and plasmid DNA; the former of which amplifi ed Aer. hydrophila,
Aer. salmonicida subsp. achromogenes, Aer. salmonicida subsp. masoucida and
atypical isolates, whereas the latter detected only Aer. salmonicida subsp. achromogenes and Aer. salmonicida subsp. salmonicida. Based on an examination of 100 ml
volumes of kidney suspension and gill swabs, the PCR detected 20 and 200 colony
forming units in 10 μl of PCR template by 16S rRNA and plasmid primers, respectively. The numbers corresponded to 10
3 and 10
4 colony forming units in 100 ml of
kidney suspension, respectively (Høie et al. 1997 ). A conclusion was reached that
the PCR detected Aer. salmonicida more often than culturing. Terminal-RFLP permitted the detection of ~30 CFU/mg of artifi cially inoculated kidney tissue (Nilsson
and Strom 2002 ). A multiplex PCR was developed for the simultaneous detection
Aer. salmonicida, Pis. salmonis, Str. phocae and V. anguillarum . The detection limit
using purifi ed total bacterial DNA was 5 pg/ μl (=5.33 × 10
4 CFU/ml). The limits of
detection using spiked tissues, i.e. kidney, liver, muscle or spleen, were
3.8 ± 0.78 × 10
3 CFU/mg (Tapia-Cammas et al. 2011 ). A possible drawback for use
of DNA probes, however, has been proposed by Hennigan et al. ( 1989 ). Using four
DNA probes in combination with seven restriction enzymes and seven strains of
Aer. salmonicida, their data suggested that the DNA sequences of the species is very
strongly conserved. They emphasised that the use of DNA probe technology to
identify different strains of Aer. salmonicida may be limited. Using reverse
transcription- multiplex PCR with primers, SV1/SV2 and SF1/SF2, which were specifi c to vapA and fstB genes, respectively, Rattanachaikunsopon and Phumkhachorn
( 2012 ) detected 10 CFU in pure culture and 30 CFU in tissue. Moreover, the method
distinguished viable from non-viable cells, and typical from atypical representatives
of Aer. salmonicida. Keeling et al. ( 2013 ) developed and validated a highly specifi c
and reproducible real-time PCR, which detected the surface array protein, vapA,
with reported sensitivity of 5 fg of DNA, 2.2 × 10
4 CFU/g of kidney tissue without
enrichment and 40 CFU/g with enrichment.
In a comparison of the sensitivity of culturing with DNA probes, the former
detected Aer. salmonicida from the kidney of only dead or moribund farmed Atlantic
salmon smolts in Ireland whereas probe technology allied to a PCR assay was capable of recognising the pathogen in water, faeces and effl uent (O’Brien et al. 1994 ).
The benefi t of species-specifi c primers and a nested PCR was demonstrated over
universal eubacterial primers when the detection limit improved from 1.4 × 10
4 CFU/
reaction to <14 CFU/sample (Taylor and Winton 2002 ). The importance of the
primer set was further highlighted in a comparative study by Byers et al. ( 2002a , b ).
Denaturing Gradient Gel Electrophoresis (DGGE) A method has been proposed and was evaluated in wild spawning coho salmon ( Oncorhynchus kisutch )
and cultured lake trout ( Salvelinus namaycush ) for the nonlethal detection of the
pathogen from mucus involved DGGE of 16S rDNA, which led to a reproducible
4-band pattern. This was distinctive from other aeromonads, and Aer. salmonicida.
The technique recognised 36 out of 52 coho salmon that were positive for Aer. salAeromonas salmonicida