223
The stress-induced furunculosis test to detect covertly infected fi sh has been very
successful, and involves intramuscular injection with corticosteroid, namely 20 mg
prednisolone acetate/kg of fi sh followed by increasing the water temperature typically from 12 to 18–20 °C. Cultures of Aer. salmonicida may be then recovered on
TSA of CBB within 3–6 days (Bullock and Stuckey 1975b ; McCarthy 1977b ; Smith
1991 ; Bullock et al. 1997 ). Using such stressed rainbow trout, culturing was the
most sensitive method for detecting Aer. salmonicida (detected 40 positives out of
80) followed by a direct FAT (detected 6 positive) and then a commercial ELISA
system (detected 6 positives) (Bullock et al. 1997 ). Overall, the culture of gill and
mucus was more sensitive (39 positives out of 80 fi sh examined) than kidney and
spleen (18 positives) (Bullock et al. 1997 ).
In the case of CE and goldfi sh ulcer disease, the pathogen appears to be located
more or less exclusively in the skin lesions. Thus, Bootsma et al. ( 1977 ) used an
inoculating wire, which was plunged below the transparent epidermis at the edge of
the ulcer, into the haemorrhagic zone. A loopful of the resulting material was
streaked onto agar media. According to Bootsma, satisfactory growth occurred on
tryptone-containing media supplemented with serum. Although the enrichment of a
culture medium by the addition of serum has been deduced as necessary for the
initial recovery of some fastidious strains, notably non-pigmented cultures, Bootsma
et al. ( 1977 ) determined that the fastidiousness of the isolates decreased during
maintenance in vitro . In our experience, strains associated with non-salmonid fi sh
are extremely diffi cult to isolate. We have greatest success with blood agar (blood
agar base [Oxoid] supplemented with 10 % v/v horse, sheep or bovine blood), which
is inoculated and incubated at 15–18 °C for up to 7 days. Even with this method,
Aer. salmonicida is recovered from only a small proportion of the clinically diseased fi sh. This begs the question about the reasons for culturability, when the
pathogen is recovered from only a proportion of obviously infected animals.
Microscopy will often reveal a greater number of bacterial cells than might be
deduced from the results of plating experiments. Perhaps, as has been argued with
L-forms (Figs. 5.9 , 5.10 ), a threshold number of bacterial cells need to be present to
enable some to be capable of producing growth in broth or on solid medium. Also,
the defi nition of growth needs to be carefully considered, insofar as the basic criterion refl ects observations with the naked eye, i.e. turbidity in broth or clearly visible
colonies. The limited growth of micro-colonies may well be missed by classical
bacteriological methods.
Characteristics of the Pathogen
Aer. salmonicida , which comprises the so-called non-motile taxon, is one of the
most important fi sh pathogens because of its widespread distribution, diverse host
range and economically devastating impact on cultivated fi sh, particularly the valuable salmonids. More has been written about Aer. salmonicida than any other bacterial fi sh pathogen. Comprehensive reviews, i.e. McCraw ( 1952 ), Herman ( 1968 ),
Aeromonas salmonicida
The stress-induced furunculosis test to detect covertly infected fi sh has been very
successful, and involves intramuscular injection with corticosteroid, namely 20 mg
prednisolone acetate/kg of fi sh followed by increasing the water temperature typically from 12 to 18–20 °C. Cultures of Aer. salmonicida may be then recovered on
TSA of CBB within 3–6 days (Bullock and Stuckey 1975b ; McCarthy 1977b ; Smith
1991 ; Bullock et al. 1997 ). Using such stressed rainbow trout, culturing was the
most sensitive method for detecting Aer. salmonicida (detected 40 positives out of
80) followed by a direct FAT (detected 6 positive) and then a commercial ELISA
system (detected 6 positives) (Bullock et al. 1997 ). Overall, the culture of gill and
mucus was more sensitive (39 positives out of 80 fi sh examined) than kidney and
spleen (18 positives) (Bullock et al. 1997 ).
In the case of CE and goldfi sh ulcer disease, the pathogen appears to be located
more or less exclusively in the skin lesions. Thus, Bootsma et al. ( 1977 ) used an
inoculating wire, which was plunged below the transparent epidermis at the edge of
the ulcer, into the haemorrhagic zone. A loopful of the resulting material was
streaked onto agar media. According to Bootsma, satisfactory growth occurred on
tryptone-containing media supplemented with serum. Although the enrichment of a
culture medium by the addition of serum has been deduced as necessary for the
initial recovery of some fastidious strains, notably non-pigmented cultures, Bootsma
et al. ( 1977 ) determined that the fastidiousness of the isolates decreased during
maintenance in vitro . In our experience, strains associated with non-salmonid fi sh
are extremely diffi cult to isolate. We have greatest success with blood agar (blood
agar base [Oxoid] supplemented with 10 % v/v horse, sheep or bovine blood), which
is inoculated and incubated at 15–18 °C for up to 7 days. Even with this method,
Aer. salmonicida is recovered from only a small proportion of the clinically diseased fi sh. This begs the question about the reasons for culturability, when the
pathogen is recovered from only a proportion of obviously infected animals.
Microscopy will often reveal a greater number of bacterial cells than might be
deduced from the results of plating experiments. Perhaps, as has been argued with
L-forms (Figs. 5.9 , 5.10 ), a threshold number of bacterial cells need to be present to
enable some to be capable of producing growth in broth or on solid medium. Also,
the defi nition of growth needs to be carefully considered, insofar as the basic criterion refl ects observations with the naked eye, i.e. turbidity in broth or clearly visible
colonies. The limited growth of micro-colonies may well be missed by classical
bacteriological methods.
Characteristics of the Pathogen
Aer. salmonicida , which comprises the so-called non-motile taxon, is one of the
most important fi sh pathogens because of its widespread distribution, diverse host
range and economically devastating impact on cultivated fi sh, particularly the valuable salmonids. More has been written about Aer. salmonicida than any other bacterial fi sh pathogen. Comprehensive reviews, i.e. McCraw ( 1952 ), Herman ( 1968 ),
Aeromonas salmonicida