89
The question concerning the necessary growth requirements for the pathogen
was addressed in a detailed study by Embley et al. ( 1982 ). These workers formulated
a rich semi-defi ned medium devoid of serum (Appendix in Chap. 12 – semi defi ned
medium), which was suitable for the cultivation of cells, but not for the initial isolation of cells from infected fi sh tissues. The semi-defi ned medium was used, however, to obtain biomass destined for lipid analyses (Embley et al. 1983 ), and inocula
for nutritional and physiological studies. Subsequently, Shieh ( 1989 a) described a
complex blood free medium, which permits the growth of the pathogen.
Characteristics of the Pathogen
At various times, the causal agent of BKD has been linked with Corynebacterium
(Ordal and Earp 1956 ; Smith 1964 ; Sanders and Fryer 1978 ; Austin and Rodgers
1980 ), Brevibacterium (Smith 1964 ), Listeria (Bullock et al. 1975 ), Lactobacillus
(Vladik et al. 1974 ) and Rickettsia (Snieszko and Griffi n 1955 ). Subsequently, it
was appreciated that the organisms were suffi ciently unique to warrant separate species status, so, Corynebacterium salmoninus was described (Sanders and Fryer
1978 ). With further information, these authors realised that the pathogen belonged
in a new, as yet undescribed genus and, therefore, proposed Renibacterium . Thus,
the causal agent of BKD became classifi ed as Renibacterium salmoninarum
(Sanders and Fryer 1980 ).
The initial diffi culties experienced in culturing the pathogen contributed signifi -
cantly to the uncertainty over its precise taxonomic status. Early work emphasised a
few morphological features, namely the presence of small (0.3–1.5 × 0.1–1.0 μm),
Gram-positive, asporogenous, non-motile, non-acid-fast rods, which frequently
occurred in pairs. Evidence of pleomorphism, metachromatic granules and a
‘coryneform’ appearance (Ordal and Earp 1956 ; Smith 1964 ) led to the initial, tenuous association with the coryneform group of bacteria, namely Corynebacterium , It
is interesting to note that the later investigation of Young and Chapman ( 1978 ) did
not substantiate the ‘coryneform’ morphology. However, transmission electron
microscopy of negatively stained cells, obtained from 28-day-old cultures on growth
medium, i.e. KDM2, revealed the presence of pleomorphism and intracellular vacuoles/granules (B. Austin, unpublished data). By using FAT on kidney smears from
coho salmon, Cvitanich ( 2004 ) observed small short rods, termed bar forms because
of their staining reaction in FAT, which could not be cultured and were not virulent.
Earp ( 1950 ) and Ordal and Earp ( 1956 ) demonstrated catalase and proteolytic
activity, and realised that there was a growth requirement for cysteine. Additional
attributes of the organism were slowly realised; in particular, Smith ( 1964 ) indicated the temperature range of growth, i.e. most rapid at 15 °C, slow at 5 and 22 °C,
and not at all at 37 °C, and determined an inability to degrade gelatin. During the
period of the late 1970s to early 1980s, a wealth of knowledge was accumulated on
Renibacterium . A low genetic diversity among North American isolates has been
indicated from multilocus enzyme electrophoresis using 44 enzymes (Starliper
1996 ). Thus, from 40 isolates, 21 electrophoretic types were recognised. Grayson
Renibacterium salmoninarum
The question concerning the necessary growth requirements for the pathogen
was addressed in a detailed study by Embley et al. ( 1982 ). These workers formulated
a rich semi-defi ned medium devoid of serum (Appendix in Chap. 12 – semi defi ned
medium), which was suitable for the cultivation of cells, but not for the initial isolation of cells from infected fi sh tissues. The semi-defi ned medium was used, however, to obtain biomass destined for lipid analyses (Embley et al. 1983 ), and inocula
for nutritional and physiological studies. Subsequently, Shieh ( 1989 a) described a
complex blood free medium, which permits the growth of the pathogen.
Characteristics of the Pathogen
At various times, the causal agent of BKD has been linked with Corynebacterium
(Ordal and Earp 1956 ; Smith 1964 ; Sanders and Fryer 1978 ; Austin and Rodgers
1980 ), Brevibacterium (Smith 1964 ), Listeria (Bullock et al. 1975 ), Lactobacillus
(Vladik et al. 1974 ) and Rickettsia (Snieszko and Griffi n 1955 ). Subsequently, it
was appreciated that the organisms were suffi ciently unique to warrant separate species status, so, Corynebacterium salmoninus was described (Sanders and Fryer
1978 ). With further information, these authors realised that the pathogen belonged
in a new, as yet undescribed genus and, therefore, proposed Renibacterium . Thus,
the causal agent of BKD became classifi ed as Renibacterium salmoninarum
(Sanders and Fryer 1980 ).
The initial diffi culties experienced in culturing the pathogen contributed signifi -
cantly to the uncertainty over its precise taxonomic status. Early work emphasised a
few morphological features, namely the presence of small (0.3–1.5 × 0.1–1.0 μm),
Gram-positive, asporogenous, non-motile, non-acid-fast rods, which frequently
occurred in pairs. Evidence of pleomorphism, metachromatic granules and a
‘coryneform’ appearance (Ordal and Earp 1956 ; Smith 1964 ) led to the initial, tenuous association with the coryneform group of bacteria, namely Corynebacterium , It
is interesting to note that the later investigation of Young and Chapman ( 1978 ) did
not substantiate the ‘coryneform’ morphology. However, transmission electron
microscopy of negatively stained cells, obtained from 28-day-old cultures on growth
medium, i.e. KDM2, revealed the presence of pleomorphism and intracellular vacuoles/granules (B. Austin, unpublished data). By using FAT on kidney smears from
coho salmon, Cvitanich ( 2004 ) observed small short rods, termed bar forms because
of their staining reaction in FAT, which could not be cultured and were not virulent.
Earp ( 1950 ) and Ordal and Earp ( 1956 ) demonstrated catalase and proteolytic
activity, and realised that there was a growth requirement for cysteine. Additional
attributes of the organism were slowly realised; in particular, Smith ( 1964 ) indicated the temperature range of growth, i.e. most rapid at 15 °C, slow at 5 and 22 °C,
and not at all at 37 °C, and determined an inability to degrade gelatin. During the
period of the late 1970s to early 1980s, a wealth of knowledge was accumulated on
Renibacterium . A low genetic diversity among North American isolates has been
indicated from multilocus enzyme electrophoresis using 44 enzymes (Starliper
1996 ). Thus, from 40 isolates, 21 electrophoretic types were recognised. Grayson
Renibacterium salmoninarum