98
Following an examination of 1239 kidney samples, Gudmundsdóttir et al. ( 1993 )
considered that a double sandwich ELISA was more sensitive than culturing on
SKDM. Yet, Bandín et al. ( 1996 ) reported the comparatively high cut-off for ELISA
of ~10
6 bacteria/g of tissue. An amount of 0.3 μg of antigen/ml was noted by Olea
et al. ( 1993 ). Sakai et al. ( 1989a , b ) favoured the indirect dot blot assay (Sakai et al.
1989a , b ; Sakai and Kobayashi 1992 ) [involving peroxidase and diaminobenzidine
tetrahydrochloride as enzyme and substrate, respectively], which detected 10
2
cells/g of kidney tissue, over iFAT, co-agglutination, microscopy by Gram-stain,
immunodiffusion and latex agglutination. Confi rmatory diagnoses were made using
dot blot and western blot assays (Sakai et al. 1990 ; see also Olivier et al. 1992 ).
Certainly, Griffi ths et al. ( 1991 ) highlighted the value of western blots over FAT and
culturing for the detection of renibacteria. Immunohistochemistry is another
approach, which is gaining popularity for the effi cient detection of BKD. In particular, the indirect peroxidase technique, as applied to tissue sections, was deemed to
be more sensitive than iFAT or Gram-staining (Hoffman et al. 1989 ). Further work
has also highlighted the value of the peroxidase-antiperoxidase immunohistochemical technique for the detection of Renibacterium (Jansson et al. 1991 ). Mucus quantitative RT-PCR was regarded as the only non-lethal system with the necessary
sensitivity and specifi city, as an alternative to the lethal approach of examining kidney (Elliott et al. 2015 ).
Whereas debate has centred over the most effective means of detecting BKD, it
may be concluded that effective diagnosis should encompass a multiplicity of methods. These include isolation and characterisation, and serology on infected tissue. It
is proposed that clinical cases of disease should be examined by FAT and culturing
methods. Asymptomatic cases should be the subject of full bacteriological
examination.
Epizootiology
To date, there has been no evidence to suggest that Renibacterium is a component
of the normal aquatic microfl ora. Indeed in one study, water and sediment from 56
fi sh farms were examined for the presence of renibacteria, but to no avail (Austin
and Rayment 1985 ). Twelve days after experimentally infecting Chinook salmon
with a high challenge dose, which led to infections with high numbers of renibacterial cells as determined by ELISA and FAT, the pathogen could be detected in the
water (McKibben and Pascho 1999 ). Survival experiments confi rmed that
Renibacterium could survive in fi sh tank sediment/faecal material for up to 21 days
in the absence of any fi sh. However, the organism was not at any time recovered
from the overlying water, suggesting that renibacteria have an affi nity with organic
matter. Longer survival times of 13 weeks in river but not ground water were
reported by Hirvelä-Koski ( 2004 ). The question regarding survival of the pathogen
in water was the topic of detailed experimentation. This confi rmed earlier work that
laboratory-grown cultures were short-lived in river water. In the absence of
3 Aerobic Gram-Positive Rods and Cocci
Following an examination of 1239 kidney samples, Gudmundsdóttir et al. ( 1993 )
considered that a double sandwich ELISA was more sensitive than culturing on
SKDM. Yet, Bandín et al. ( 1996 ) reported the comparatively high cut-off for ELISA
of ~10
6 bacteria/g of tissue. An amount of 0.3 μg of antigen/ml was noted by Olea
et al. ( 1993 ). Sakai et al. ( 1989a , b ) favoured the indirect dot blot assay (Sakai et al.
1989a , b ; Sakai and Kobayashi 1992 ) [involving peroxidase and diaminobenzidine
tetrahydrochloride as enzyme and substrate, respectively], which detected 10
2
cells/g of kidney tissue, over iFAT, co-agglutination, microscopy by Gram-stain,
immunodiffusion and latex agglutination. Confi rmatory diagnoses were made using
dot blot and western blot assays (Sakai et al. 1990 ; see also Olivier et al. 1992 ).
Certainly, Griffi ths et al. ( 1991 ) highlighted the value of western blots over FAT and
culturing for the detection of renibacteria. Immunohistochemistry is another
approach, which is gaining popularity for the effi cient detection of BKD. In particular, the indirect peroxidase technique, as applied to tissue sections, was deemed to
be more sensitive than iFAT or Gram-staining (Hoffman et al. 1989 ). Further work
has also highlighted the value of the peroxidase-antiperoxidase immunohistochemical technique for the detection of Renibacterium (Jansson et al. 1991 ). Mucus quantitative RT-PCR was regarded as the only non-lethal system with the necessary
sensitivity and specifi city, as an alternative to the lethal approach of examining kidney (Elliott et al. 2015 ).
Whereas debate has centred over the most effective means of detecting BKD, it
may be concluded that effective diagnosis should encompass a multiplicity of methods. These include isolation and characterisation, and serology on infected tissue. It
is proposed that clinical cases of disease should be examined by FAT and culturing
methods. Asymptomatic cases should be the subject of full bacteriological
examination.
Epizootiology
To date, there has been no evidence to suggest that Renibacterium is a component
of the normal aquatic microfl ora. Indeed in one study, water and sediment from 56
fi sh farms were examined for the presence of renibacteria, but to no avail (Austin
and Rayment 1985 ). Twelve days after experimentally infecting Chinook salmon
with a high challenge dose, which led to infections with high numbers of renibacterial cells as determined by ELISA and FAT, the pathogen could be detected in the
water (McKibben and Pascho 1999 ). Survival experiments confi rmed that
Renibacterium could survive in fi sh tank sediment/faecal material for up to 21 days
in the absence of any fi sh. However, the organism was not at any time recovered
from the overlying water, suggesting that renibacteria have an affi nity with organic
matter. Longer survival times of 13 weeks in river but not ground water were
reported by Hirvelä-Koski ( 2004 ). The question regarding survival of the pathogen
in water was the topic of detailed experimentation. This confi rmed earlier work that
laboratory-grown cultures were short-lived in river water. In the absence of
3 Aerobic Gram-Positive Rods and Cocci