427
spleen and 5 cells/g of ovarian fl uid (Baliarda et al. 2002 ). Ovarian fl uid eggs and
gill washings and benthic diatoms were used with a nested PCR by Taylor ( 2004 )
and Izumi et al. ( 2005 ), respectively, with again commendable results. A Taq Manbased PCR used primers amplifying a 971 bp fragment of the 16S rRNA, and
detected 1.1 pg of Fla. psychrophilum DNA, which equated with 4.7 CFU/PCR
reaction (del Cerro et al. 2002). Terminal-RFLP permitted the detection of ~30 CFU
of Fla. psychrophilum /mg of artifi cially inoculated kidney tissue (Nilsson and
Strom 2002 ). 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
5 CFU/reaction to <14 CFU/sample (Taylor and Winton 2002 ). A specifi c
and rapid LAMP assay, which takes only 70 min to complete, has been proposed for
the detection of Fla. psychrophilum with detection in the range of 2.0 × 10
1 to
2.0 × 10
9 DNA copies/reaction (Fujiwara-Nagata and Eguchi 2009 ). A highly specifi c quantitative PCR was published, and stated to be effective in detecting only
two bacteria/reaction, which corresponded to 800 bacterial cells/fi sh or 20 fl avobacterial cells/mg of tissue (Orieux et al. 2011 ). A RT-PCR involving a sequence that
encodes a hypothetical protein of unknown function had a detection limit of 3.1
genome equivalents/reaction, and successfully recognized Fla. psychrophilum in
naturally occurring outbreaks of disease on rainbow trout farms (Marancik and
Wiens 2013 ). In a comparison of PCR methods, Suzuki et al. ( 2011 ) identifi ed
issues with false positives when targeting DNA gyrase subunit gene gyrA but not so
for gyrB and peptidyl-prolyl cis-trans isomerase C gene ppiC. However, the PCR
targeting 16S rDNA was more sensitive. An ideal situation would involve techniques that could recognise and differentiate between multiple diseases, and this has
been achieved with multiplex PCR. Del Cerro et al. (2002) detected simultaneously
Aer. salmonicida, Fla. psychrophilum and Y. ruckeri in fi sh tissues , recognising the
equivalent of 6, 0.6 and 27 CFU, respectively. A highly sensitive and specifi c quantitative RT-PCR based on the single copy gene ß DNA-dependent RNA polymerase
revealed a detection limit of 20 gene copies and a quantifi cation limit of 10
3 gene
copies/reaction (Streppaeava et al. 2014). High-gradient immunomagnetic separation with a PCR was regarded as more sensitive than PCR alone, and permitted the
recording of results in <3.5 h (Ryumae et al. 2012 ).
Which Method is Best? A comparison was made of the sensitivities of various
methods to detect the pathogen in tissue and ovarian fl uid, which were collected
from 224 fi sh at fi ve hatcheries. The methods used included ELISA, a membranefi ltration FAT, culture and nested PCR, with the fi rst mentioned being regarded as
the appropriate technique for the screening of broodstock, and for indicating the
severity of infection (Long et al. 2012 ).
Flavobacterium psychrophilum
spleen and 5 cells/g of ovarian fl uid (Baliarda et al. 2002 ). Ovarian fl uid eggs and
gill washings and benthic diatoms were used with a nested PCR by Taylor ( 2004 )
and Izumi et al. ( 2005 ), respectively, with again commendable results. A Taq Manbased PCR used primers amplifying a 971 bp fragment of the 16S rRNA, and
detected 1.1 pg of Fla. psychrophilum DNA, which equated with 4.7 CFU/PCR
reaction (del Cerro et al. 2002). Terminal-RFLP permitted the detection of ~30 CFU
of Fla. psychrophilum /mg of artifi cially inoculated kidney tissue (Nilsson and
Strom 2002 ). 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
5 CFU/reaction to <14 CFU/sample (Taylor and Winton 2002 ). A specifi c
and rapid LAMP assay, which takes only 70 min to complete, has been proposed for
the detection of Fla. psychrophilum with detection in the range of 2.0 × 10
1 to
2.0 × 10
9 DNA copies/reaction (Fujiwara-Nagata and Eguchi 2009 ). A highly specifi c quantitative PCR was published, and stated to be effective in detecting only
two bacteria/reaction, which corresponded to 800 bacterial cells/fi sh or 20 fl avobacterial cells/mg of tissue (Orieux et al. 2011 ). A RT-PCR involving a sequence that
encodes a hypothetical protein of unknown function had a detection limit of 3.1
genome equivalents/reaction, and successfully recognized Fla. psychrophilum in
naturally occurring outbreaks of disease on rainbow trout farms (Marancik and
Wiens 2013 ). In a comparison of PCR methods, Suzuki et al. ( 2011 ) identifi ed
issues with false positives when targeting DNA gyrase subunit gene gyrA but not so
for gyrB and peptidyl-prolyl cis-trans isomerase C gene ppiC. However, the PCR
targeting 16S rDNA was more sensitive. An ideal situation would involve techniques that could recognise and differentiate between multiple diseases, and this has
been achieved with multiplex PCR. Del Cerro et al. (2002) detected simultaneously
Aer. salmonicida, Fla. psychrophilum and Y. ruckeri in fi sh tissues , recognising the
equivalent of 6, 0.6 and 27 CFU, respectively. A highly sensitive and specifi c quantitative RT-PCR based on the single copy gene ß DNA-dependent RNA polymerase
revealed a detection limit of 20 gene copies and a quantifi cation limit of 10
3 gene
copies/reaction (Streppaeava et al. 2014). High-gradient immunomagnetic separation with a PCR was regarded as more sensitive than PCR alone, and permitted the
recording of results in <3.5 h (Ryumae et al. 2012 ).
Which Method is Best? A comparison was made of the sensitivities of various
methods to detect the pathogen in tissue and ovarian fl uid, which were collected
from 224 fi sh at fi ve hatcheries. The methods used included ELISA, a membranefi ltration FAT, culture and nested PCR, with the fi rst mentioned being regarded as
the appropriate technique for the screening of broodstock, and for indicating the
severity of infection (Long et al. 2012 ).
Flavobacterium psychrophilum