267
epizootics, were of the aggregating type. From the results of experiments, Udey and
Fryer ( 1978 ) concluded that the presence of the A-layer was necessary for virulence. However, they contended that more work was needed to establish whether or
not the A-layer alone could confer virulence. The discovery of the A-layer generated much interest, resulting in further study of its chemical composition and its
specifi c role in fi sh pathology. Kay et al. ( 1981 ) succeeded in purifying the A-layer
from virulent isolates, and concluded that it was composed of a surface-localised
protein with a molecular weight of 49 kDa. Phipps et al. ( 1983 ) continued with
work on purifi cation and characterisation of the substance, determining that it was
hydrophobic in nature, present on the entire cell surface, did not possess any enzymic activity, but instead constituted a macromolecular refractive protein barrier
which was essential for virulence. Meanwhile, an independent parallel investigation
of Evenberg et al. ( 1982 ) highlighted the relationship between auto-agglutination
and the presence of the A-layer. This group examined the cell envelope protein patterns of a variety of isolates obtained from a wide range of geographical locations
and different fi sh species (i.e. carp, minnow, goldfi sh and salmonids). These fi sh
were suffering from either furunculosis, CE or ulcer disease. A major protein
(molecular weight = 54 kDa) was found in all auto-aggregating strains, but little or
no trace occurred in isolates that were not auto-agglutinating. When examinations
for the presence of the protein were carried out after a change of growth medium,
i.e. replacement of horse serum by synthetic sea salt, it was observed that an almost
complete loss of the additional cell envelope and the auto-agglutinating ability of
the isolate had occurred. Using gel immunoradio assays, it was also determined that
the extra cell envelope proteins of all the isolates, irrespective of fi sh host, type of
infection or geographical source, were immunologically related. Evenberg and
Lugtenberg ( 1982 ) pursued this topic, and described the protein as water insoluble
with an amino acid composition similar to those of the additional surface layers of
other bacteria, e.g. the adhesive K88 fi mbriae of enteropathogenic strains of Esch.
coli . It is particularly relevant that the fi ndings of Evenberg et al. ( 1982 ), concerning
the auto-agglutinating ability of ‘atypical’ strains from cases of CE and ulcer disease, and the presence of the A-layer, were in excellent agreement with the work of
Trust et al. ( 1980b , c ) and Hamilton et al. ( 1981 ). These earlier studies deduced the
presence of an outer layer protein, which was estimated to have a molecular weight
of 50 kDa. Evidence was provided by Ishiguro et al. ( 1981 ) that loss of the A-layer
and loss of auto-agglutinating properties resulted in decreased virulence. After
examining the effects of temperatures on the growth of Aer. salmonicida, it was
shown that in cells, cultured at 30 °C (the generally accepted upper limit for the
organism), virulence was restricted to <10 % of the population. The avirulent attenuated cells that resulted from use of the higher growth temperature, did not autoagglutinate and, for that matter, did not possess the A-layer. It is interesting to note
that higher maximum growth temperatures were recorded for the attenuated strains,
in comparison to their virulent counterparts. Perhaps, this is explained by their
selection at high temperatures. Because of this observation, Ishiguro et al. ( 1981 )
hypothesised that the A-layer is important in determining physical properties of the
cell envelope, and that these properties undergo a change when the A-layer is lost,
Aeromonas salmonicida
epizootics, were of the aggregating type. From the results of experiments, Udey and
Fryer ( 1978 ) concluded that the presence of the A-layer was necessary for virulence. However, they contended that more work was needed to establish whether or
not the A-layer alone could confer virulence. The discovery of the A-layer generated much interest, resulting in further study of its chemical composition and its
specifi c role in fi sh pathology. Kay et al. ( 1981 ) succeeded in purifying the A-layer
from virulent isolates, and concluded that it was composed of a surface-localised
protein with a molecular weight of 49 kDa. Phipps et al. ( 1983 ) continued with
work on purifi cation and characterisation of the substance, determining that it was
hydrophobic in nature, present on the entire cell surface, did not possess any enzymic activity, but instead constituted a macromolecular refractive protein barrier
which was essential for virulence. Meanwhile, an independent parallel investigation
of Evenberg et al. ( 1982 ) highlighted the relationship between auto-agglutination
and the presence of the A-layer. This group examined the cell envelope protein patterns of a variety of isolates obtained from a wide range of geographical locations
and different fi sh species (i.e. carp, minnow, goldfi sh and salmonids). These fi sh
were suffering from either furunculosis, CE or ulcer disease. A major protein
(molecular weight = 54 kDa) was found in all auto-aggregating strains, but little or
no trace occurred in isolates that were not auto-agglutinating. When examinations
for the presence of the protein were carried out after a change of growth medium,
i.e. replacement of horse serum by synthetic sea salt, it was observed that an almost
complete loss of the additional cell envelope and the auto-agglutinating ability of
the isolate had occurred. Using gel immunoradio assays, it was also determined that
the extra cell envelope proteins of all the isolates, irrespective of fi sh host, type of
infection or geographical source, were immunologically related. Evenberg and
Lugtenberg ( 1982 ) pursued this topic, and described the protein as water insoluble
with an amino acid composition similar to those of the additional surface layers of
other bacteria, e.g. the adhesive K88 fi mbriae of enteropathogenic strains of Esch.
coli . It is particularly relevant that the fi ndings of Evenberg et al. ( 1982 ), concerning
the auto-agglutinating ability of ‘atypical’ strains from cases of CE and ulcer disease, and the presence of the A-layer, were in excellent agreement with the work of
Trust et al. ( 1980b , c ) and Hamilton et al. ( 1981 ). These earlier studies deduced the
presence of an outer layer protein, which was estimated to have a molecular weight
of 50 kDa. Evidence was provided by Ishiguro et al. ( 1981 ) that loss of the A-layer
and loss of auto-agglutinating properties resulted in decreased virulence. After
examining the effects of temperatures on the growth of Aer. salmonicida, it was
shown that in cells, cultured at 30 °C (the generally accepted upper limit for the
organism), virulence was restricted to <10 % of the population. The avirulent attenuated cells that resulted from use of the higher growth temperature, did not autoagglutinate and, for that matter, did not possess the A-layer. It is interesting to note
that higher maximum growth temperatures were recorded for the attenuated strains,
in comparison to their virulent counterparts. Perhaps, this is explained by their
selection at high temperatures. Because of this observation, Ishiguro et al. ( 1981 )
hypothesised that the A-layer is important in determining physical properties of the
cell envelope, and that these properties undergo a change when the A-layer is lost,
Aeromonas salmonicida