253
tion of the ignorance of the parameters necessary for their recovery, as the occurrence
of a truly non-culturable, specialised survival state. Subsequently by means of fl ow
cytometry with rhodamine 123, they established a NCBV state in sterile lake water
(Morgan et al. 1993 ). However, fl ow cytometry indicated that cellular properties
related with viability was lost shortly after culturability disappeared in distilled
water, but not so in lake water (Deere et al. 1996b ). Additionally, these workers
marked an isolate of Aer. salmonicida with the xylE gene, using the plasmid
pLV1013. This isolate was culturable on TSA from normal (non sterile) lake water
for 3 weeks, after which non-culturability developed, with the NCBV cells retaining
chromosomal and plasmid DNA. The NCBV state can be postponed (60 days was
mentioned by Pickup et al. 1996 ) by the addition of high levels of nutrient, especially 125 μM quantities of the amino acids arginine and methionine, to experimental microcosms. Aer. salmonicida decreased in size and became rounded, but were
still culturable (Pickup et al. 1996 ).
The development of a dormant, nonculturable state of Aer. salmonicida in seawater at 4 °C was apparent from the work of Ferguson et al. ( 1995 ). These workers
incorporated a luciferase gene, luxAB , from V. fi scheri into Aer. salmonicida and
followed the fate of the cells. As before, intact nonculturable cells could not be
resurrected.
There has always been a dilemma about the relevance of cells that cannot be
cultured, to fi sh. It is worth heeding the results of Stanley et al. ( 2002 ), who determined that only culturable cells in laboratory microcosms could induce furunculosis
upon injection into fi sh, and not PCR or ELISA positive samples which could not
be supported by culturing evidence.
Ecology of Aeromonas salmonicida – An Explanation To develop some previous
points, there is tentative evidence to support the possibility that Aer. salmonicida
undergoes suffi cient modifi cations to its morphology in seawater so as to be only
recoverable on specialised media. Thus while conducting experiments on the
survival of Aer. salmonicida in seawater, Effendi and Austin ( 1991 ) found that samples where the pathogen was believed to be absent (or unculturable) actually contained cells which passed through 0.22 and 0.45 μm pore size porosity fi lters. These
isolates grew on specialised media designed for the recovery of L-forms (Fig. 5.8 ),
and showed agreement with the characteristics of Aer. salmonicida L-forms as
reported by McIntosh and Austin ( 1988 , 1990 , 1991b ). Subsequently, Aer. salmonicida colonies developed on basal marine agar (BMA) plates inoculated with material from turbid L-form broth medium. On this basis, Effendi and Austin ( 1991 )
recorded populations of ca. 10
3 Aer. salmonicida cells/ml in the microcosms after
corresponding enumeration of colonies on BMA had reached zero. Thus, they suggested that the existence of specialised forms, e.g. L-forms, of Aer. salmonicida,
may be a factor in the diffi culties previous researchers have experienced in attempts
to recover the pathogen from environmental samples. Continuing this theme,
Effendi and Austin ( 1995a ) examined the characteristics of the so-called NCBV
cells. Using a marine microcosm, it was observed that these NCBV cells became
much smaller and coccoid while retaining respiratory activity as measured by the
Aeromonas salmonicida
tion of the ignorance of the parameters necessary for their recovery, as the occurrence
of a truly non-culturable, specialised survival state. Subsequently by means of fl ow
cytometry with rhodamine 123, they established a NCBV state in sterile lake water
(Morgan et al. 1993 ). However, fl ow cytometry indicated that cellular properties
related with viability was lost shortly after culturability disappeared in distilled
water, but not so in lake water (Deere et al. 1996b ). Additionally, these workers
marked an isolate of Aer. salmonicida with the xylE gene, using the plasmid
pLV1013. This isolate was culturable on TSA from normal (non sterile) lake water
for 3 weeks, after which non-culturability developed, with the NCBV cells retaining
chromosomal and plasmid DNA. The NCBV state can be postponed (60 days was
mentioned by Pickup et al. 1996 ) by the addition of high levels of nutrient, especially 125 μM quantities of the amino acids arginine and methionine, to experimental microcosms. Aer. salmonicida decreased in size and became rounded, but were
still culturable (Pickup et al. 1996 ).
The development of a dormant, nonculturable state of Aer. salmonicida in seawater at 4 °C was apparent from the work of Ferguson et al. ( 1995 ). These workers
incorporated a luciferase gene, luxAB , from V. fi scheri into Aer. salmonicida and
followed the fate of the cells. As before, intact nonculturable cells could not be
resurrected.
There has always been a dilemma about the relevance of cells that cannot be
cultured, to fi sh. It is worth heeding the results of Stanley et al. ( 2002 ), who determined that only culturable cells in laboratory microcosms could induce furunculosis
upon injection into fi sh, and not PCR or ELISA positive samples which could not
be supported by culturing evidence.
Ecology of Aeromonas salmonicida – An Explanation To develop some previous
points, there is tentative evidence to support the possibility that Aer. salmonicida
undergoes suffi cient modifi cations to its morphology in seawater so as to be only
recoverable on specialised media. Thus while conducting experiments on the
survival of Aer. salmonicida in seawater, Effendi and Austin ( 1991 ) found that samples where the pathogen was believed to be absent (or unculturable) actually contained cells which passed through 0.22 and 0.45 μm pore size porosity fi lters. These
isolates grew on specialised media designed for the recovery of L-forms (Fig. 5.8 ),
and showed agreement with the characteristics of Aer. salmonicida L-forms as
reported by McIntosh and Austin ( 1988 , 1990 , 1991b ). Subsequently, Aer. salmonicida colonies developed on basal marine agar (BMA) plates inoculated with material from turbid L-form broth medium. On this basis, Effendi and Austin ( 1991 )
recorded populations of ca. 10
3 Aer. salmonicida cells/ml in the microcosms after
corresponding enumeration of colonies on BMA had reached zero. Thus, they suggested that the existence of specialised forms, e.g. L-forms, of Aer. salmonicida,
may be a factor in the diffi culties previous researchers have experienced in attempts
to recover the pathogen from environmental samples. Continuing this theme,
Effendi and Austin ( 1995a ) examined the characteristics of the so-called NCBV
cells. Using a marine microcosm, it was observed that these NCBV cells became
much smaller and coccoid while retaining respiratory activity as measured by the
Aeromonas salmonicida