426
Diagnosis
Phenotypic Methods Congo red has been used successfully to differentiate Fla.
psychrophilum from other fl avobacteria (Crump and Kay 2008 ). For this, 100 μg/ml
of Congo red was incorporated into tryptone yeast extract salt (TYES) medium, and
inoculated with a culture with incubation at 15
o- C for 4-days. Fla. psychrophilum
was inhibited by Congo red, and did not grow on the medium, whereas other fl avobacteria grew well (Crump and Kay 2008 ).
Serology Pacha ( 1968 ) implied that whole-cell agglutination reactions were effective for differentiating Fla. psychrophilum from other (unnamed) myxobacteria.
Also together with ELISA, the approach allowed a useable typing scheme to be
devised (Mata et al. 2002 ). Rapid identifi cation of Fla. psychrophilum was achieved
by ELISA, which detected ≥1 × 10
4 cells/ml from infected spleen (Rangdale and
Way 1995 ). Indeed, a detection level of 1.6 × 10
3 CFUs was reported in spiked kidney homogenates (Lindstrom et al. 2009 ). By use of ELISA, a useful typing scheme
was devised for Fla. psychrophilum , recognising 7 serogroups with host specifi city
(Mata et al. 2002 ). Lorenzen and Karas ( 1992 ) detailed an immunofl uorescence
technique for detecting Fla. psychrophilum in the spleen of rainbow trout suffering
with RTFS.
Immunomagnetic separation and fl ow cytometry was developed, and involved
use of fl uorescent magnetic beads and 5-cyano-2,3-ditolyl tetrazolium chloride. The
pathogen was harvested using fl uorescent magnetic beads with a single antigenantibody reaction. Then, the tetrazolium dye developed into a red fl uorescent
coloured formazan that was detectable by fl ow cytometry. In use, it took <3 h to
achieve a result (Hibi et al. 2012 ).
Molecular Methods A PCR was sensitive enough to detect 1.5 CFU/PCR reaction
tube of Fla. psychrophilum from apparently healthy coho salmon eggs and juvenile
ayu (Izumi and Wakabayashi 1997 ). In situ hybridization using digoxigenin-labelled
16S rDNA probes led to the detection of Fla. psychrophilum in experimentally
(after immersion or subcutaneous injection – in gills, heart, kidney, muscle and
spleen, but not in the brain, intestine, liver, pyloric caeca or stomach) and naturally
(intestine, liver, pancreas, pyloric caeca and stomach) infected ayu (Liu et al. 2001 ).
Similarly, PCR technology has proven to be successful for the detection of Fla.
psychrophilum and T. maritimum , and was considered to be more sensitive than
culturing (Wiklund et al. 2000 ; Avendaño-Herrera et al. 2004a ). In experiments
using brain tissue seeded with the pathogen, the detection limit was 0.4 CFU/PCR
tube, which corresponded to 17 CFU/g of brain tissue. Using normal freshwater, i.e.
containing a mixed microbial population, seeded with Fla. psychrophilum , the
detection limit was 1.7 CFU/PCR tube, which corresponded to 110 CFU/ml of
water. The PCR detected the pathogen in water from a fi sh farm, whereas culturing
did not reveal the presence of the organism (Wiklund et al. 2000 ). A nested PCR
enabled a detection limit of 1 cell/PCR tube, which was equivalent to 10 cells/g of
7 Flavobacteria and Cytophagas
Diagnosis
Phenotypic Methods Congo red has been used successfully to differentiate Fla.
psychrophilum from other fl avobacteria (Crump and Kay 2008 ). For this, 100 μg/ml
of Congo red was incorporated into tryptone yeast extract salt (TYES) medium, and
inoculated with a culture with incubation at 15
o- C for 4-days. Fla. psychrophilum
was inhibited by Congo red, and did not grow on the medium, whereas other fl avobacteria grew well (Crump and Kay 2008 ).
Serology Pacha ( 1968 ) implied that whole-cell agglutination reactions were effective for differentiating Fla. psychrophilum from other (unnamed) myxobacteria.
Also together with ELISA, the approach allowed a useable typing scheme to be
devised (Mata et al. 2002 ). Rapid identifi cation of Fla. psychrophilum was achieved
by ELISA, which detected ≥1 × 10
4 cells/ml from infected spleen (Rangdale and
Way 1995 ). Indeed, a detection level of 1.6 × 10
3 CFUs was reported in spiked kidney homogenates (Lindstrom et al. 2009 ). By use of ELISA, a useful typing scheme
was devised for Fla. psychrophilum , recognising 7 serogroups with host specifi city
(Mata et al. 2002 ). Lorenzen and Karas ( 1992 ) detailed an immunofl uorescence
technique for detecting Fla. psychrophilum in the spleen of rainbow trout suffering
with RTFS.
Immunomagnetic separation and fl ow cytometry was developed, and involved
use of fl uorescent magnetic beads and 5-cyano-2,3-ditolyl tetrazolium chloride. The
pathogen was harvested using fl uorescent magnetic beads with a single antigenantibody reaction. Then, the tetrazolium dye developed into a red fl uorescent
coloured formazan that was detectable by fl ow cytometry. In use, it took <3 h to
achieve a result (Hibi et al. 2012 ).
Molecular Methods A PCR was sensitive enough to detect 1.5 CFU/PCR reaction
tube of Fla. psychrophilum from apparently healthy coho salmon eggs and juvenile
ayu (Izumi and Wakabayashi 1997 ). In situ hybridization using digoxigenin-labelled
16S rDNA probes led to the detection of Fla. psychrophilum in experimentally
(after immersion or subcutaneous injection – in gills, heart, kidney, muscle and
spleen, but not in the brain, intestine, liver, pyloric caeca or stomach) and naturally
(intestine, liver, pancreas, pyloric caeca and stomach) infected ayu (Liu et al. 2001 ).
Similarly, PCR technology has proven to be successful for the detection of Fla.
psychrophilum and T. maritimum , and was considered to be more sensitive than
culturing (Wiklund et al. 2000 ; Avendaño-Herrera et al. 2004a ). In experiments
using brain tissue seeded with the pathogen, the detection limit was 0.4 CFU/PCR
tube, which corresponded to 17 CFU/g of brain tissue. Using normal freshwater, i.e.
containing a mixed microbial population, seeded with Fla. psychrophilum , the
detection limit was 1.7 CFU/PCR tube, which corresponded to 110 CFU/ml of
water. The PCR detected the pathogen in water from a fi sh farm, whereas culturing
did not reveal the presence of the organism (Wiklund et al. 2000 ). A nested PCR
enabled a detection limit of 1 cell/PCR tube, which was equivalent to 10 cells/g of
7 Flavobacteria and Cytophagas