245
umn. Similarly, using an oxidase-negative atypical isolate, sterilised microcosms
and culturing techniques, Wiklund ( 1995a ) deduced that survival was better at 4 °C
than 15 °C in brackish rather than sea or fresh water, and in the presence of particulates, i.e. sand. The addition of nutrients did not resuscitate cells after colony counts
declined to zero. This is important, insofar as workers have been generally unsuccessful at retrieving culturable cells after plate counts declined to zero, regardless of
the method that indicated cells or cellular components remained.
McCarthy attributed the discrepancies among the various investigations to the
technical diffi culty of isolating Aer. salmonicida from mixed cultures. The temperature at which the experiments were run may well have also infl uenced the results.
For instance, McCarthy ( 1980 ) conducted his experiments between 11 and 13 °C,
and reported longer survival times for the pathogen in fresh, brackish and seawater
(17, 24 and 8 days, respectively), than had been recorded by most other investigators (Table 5.2 ). Also, the differences may refl ect inherent variations between
cultures.
In the majority of reports about the survival of Aer. salmonicida, a large initial
inoculum of the bacterium, usually 10
6 to 10
7 cells/ml of sample, was used. It is
unlikely, however, that the pathogen would occur in these numbers even in the event
of a free-living existence in the natural environment, except perhaps during epizootics where moribund and dead fi sh were releasing large numbers of Aer. salmonicida
into the immediate vicinity. Therefore, studies were undertaken using a low number
of cells, ca. 10
1 to 10
2
/ml, as an inoculum (Allen 1982 ). When the pathogen was
placed in sterilised reservoir water in such minimal numbers, and incubated at
15 °C, the organism underwent a severe reduction in numbers within 72 h, such as
to be virtually unrecoverable by plating methods on solid non-selective medium.
From these results, which are in contrast to other studies, it becomes apparent that
several factors, including the size of the inoculum and the temperature at which the
experiments are conducted, are crucial in determining the outcome of survival studies. In contrast, when an inoculum of approximately 10
5 to 10
6 cells/ml was placed
into sterilised reservoir water, Aer. salmonicida survived and multiplied with up to
10
8 cells/ml, in the system in 72 h. It could still be recovered in substantial numbers
(10
7 cells/ml) at 55 days, when the experiment was concluded. In addition, nutrient
conditions appeared to have an effect as it was observed that supplementation with
low concentrations of nutrient, e.g. 0.005 % (w/v) brain heart infusion broth, caused
an increase in the number of Aer. salmonicida cells within 24 h. This increase was
maintained until the end of the sampling period. The addition of nutrient, moreover,
caused the increase in numbers of the pathogen regardless of whether the initial
inoculum of cells was large or small. Supplementation with nutrient was also
reported to increase survival time by McCraw ( 1952 ), who observed that the addition of 0.1 % (w/v) peptone to seawater enabled Aer. salmonicida to survive up to 80
days, a much longer time than recorded for unsupplemented sea water.
The ability of Aer. salmonicida to persist in mud (sediment) or detritus in the fi sh
farm environment has also been examined. McCarthy ( 1980 ) demonstrated that the
pathogen was able to survive in numbers of ca. 10
5 viable cells in fi sh pond mud and
Aeromonas salmonicida
umn. Similarly, using an oxidase-negative atypical isolate, sterilised microcosms
and culturing techniques, Wiklund ( 1995a ) deduced that survival was better at 4 °C
than 15 °C in brackish rather than sea or fresh water, and in the presence of particulates, i.e. sand. The addition of nutrients did not resuscitate cells after colony counts
declined to zero. This is important, insofar as workers have been generally unsuccessful at retrieving culturable cells after plate counts declined to zero, regardless of
the method that indicated cells or cellular components remained.
McCarthy attributed the discrepancies among the various investigations to the
technical diffi culty of isolating Aer. salmonicida from mixed cultures. The temperature at which the experiments were run may well have also infl uenced the results.
For instance, McCarthy ( 1980 ) conducted his experiments between 11 and 13 °C,
and reported longer survival times for the pathogen in fresh, brackish and seawater
(17, 24 and 8 days, respectively), than had been recorded by most other investigators (Table 5.2 ). Also, the differences may refl ect inherent variations between
cultures.
In the majority of reports about the survival of Aer. salmonicida, a large initial
inoculum of the bacterium, usually 10
6 to 10
7 cells/ml of sample, was used. It is
unlikely, however, that the pathogen would occur in these numbers even in the event
of a free-living existence in the natural environment, except perhaps during epizootics where moribund and dead fi sh were releasing large numbers of Aer. salmonicida
into the immediate vicinity. Therefore, studies were undertaken using a low number
of cells, ca. 10
1 to 10
2
/ml, as an inoculum (Allen 1982 ). When the pathogen was
placed in sterilised reservoir water in such minimal numbers, and incubated at
15 °C, the organism underwent a severe reduction in numbers within 72 h, such as
to be virtually unrecoverable by plating methods on solid non-selective medium.
From these results, which are in contrast to other studies, it becomes apparent that
several factors, including the size of the inoculum and the temperature at which the
experiments are conducted, are crucial in determining the outcome of survival studies. In contrast, when an inoculum of approximately 10
5 to 10
6 cells/ml was placed
into sterilised reservoir water, Aer. salmonicida survived and multiplied with up to
10
8 cells/ml, in the system in 72 h. It could still be recovered in substantial numbers
(10
7 cells/ml) at 55 days, when the experiment was concluded. In addition, nutrient
conditions appeared to have an effect as it was observed that supplementation with
low concentrations of nutrient, e.g. 0.005 % (w/v) brain heart infusion broth, caused
an increase in the number of Aer. salmonicida cells within 24 h. This increase was
maintained until the end of the sampling period. The addition of nutrient, moreover,
caused the increase in numbers of the pathogen regardless of whether the initial
inoculum of cells was large or small. Supplementation with nutrient was also
reported to increase survival time by McCraw ( 1952 ), who observed that the addition of 0.1 % (w/v) peptone to seawater enabled Aer. salmonicida to survive up to 80
days, a much longer time than recorded for unsupplemented sea water.
The ability of Aer. salmonicida to persist in mud (sediment) or detritus in the fi sh
farm environment has also been examined. McCarthy ( 1980 ) demonstrated that the
pathogen was able to survive in numbers of ca. 10
5 viable cells in fi sh pond mud and
Aeromonas salmonicida