266
The A-Layer
The A-layer is now thought to be the product of a single chromosomal gene (Belland
and Trust 1985 ), is produced in vivo (Ellis et al. 1997 ) and contributes to survival in
macrophages (Daly et al. 1996 ). The virulence array protein gene A ( vapA ) , which
encodes the A-protein has been sequenced, and differences noted in the amino acids
between typical and atypical isolates, with homogeneity among the former but heterogeneity with the latter. These differences undoubtedly lead to antigenic differences among atypical isolates (Lund and Mikkelsen 2004 ). First reported by Udey
and Fryer ( 1978 ), and resulting from detailed electron microscopic studies, the
A-layer was determined to be correlated with virulence (e.g. Madetoja et al. 2003 );
an insulin-binding capacity for the A-layer has been documented (Nisr et al. 2012 ).
It was observed that virulent strains possessed the A-layer, whereas avirulent isolates did not. In addition, the presence of the A-layer was found to correspond with
strong auto-agglutinating properties of the organism, and to the adhesion to fi sh
tissue culture cells. The auto-agglutination trait has been found to be infl uenced by
temperature, with weak and strong auto-agglutination at 25 and 15–20 °C, respectively (Moki et al. 1995 ). The presence of the A-layer may confer protection against
phagocytosis and thus destruction by macrophages (Olivier et al. 1986 ; Graham
et al. 1988 ). Essentially, these workers noted that avirulent cells, i.e. those without
an A-layer, were phagocytosed and destroyed when virulent cells with A-layer were
more resistant. Moreover, the bacteriocidal activity of macrophages was stimulated
by prior exposure to low doses of Ren. salmoninarum, but inhibited by high amounts
of living or dead renibacterial cells or the p57 antigen (Siegel and Congleton 1997 ).
Interestingly, it was deduced that living and formalised virulent cells, in the absence
of serum, attracted macrophages more readily than avirulent cells after a period of
90 min (Weeks-Perkins and Ellis 1995 ). The surface layer may inhibit growth at
30 °C, enhance cell fi lamentation at 37 °C, and enhance uptake of the hydrophobic
antibiotics streptonigrin and chloramphenicol (Garduño et al. 1994 ). Following the
intravenous injection of purifi ed A-layer protein into Atlantic salmon, the protein
located to the epithelial cells in renal proximal tubules of the head kidney (Stensvåg
et al. 1999 ).
For its formation, Belland and Trust ( 1985 ) reasoned that the A-layer subunits
pass though the periplasm and across the outer membrane for assembly on the cell
surface. A requirement for the presence of O-polysaccharide chains, for which the
AbcA protein is involved in biosynthesis (Noonan and Trust 1995 ) on the LPS was
reported as necessary for the assembly of A-layer (Dooley et al. 1989 ). These virulent, auto-agglutinating forms produce characteristic deep blue colonies on CBB
agar (Wilson and Horne 1986 ; Bernoth 1990 ). Sakai ( 1986a , b ) postulated that a
possible mechanism for auto-agglutination and adhesion could be attributed to the
presence of net negative electrical charge in the interiors or on the surfaces of cells.
In particular, pathogenic cultures were highly adhesive (Sakai 1987 ). It should be
emphasised that Udey and Fryer ( 1978 ) determined that strains maintained for long
periods in laboratory conditions were not auto-agglutinating, and demonstrated
reduced virulence. Conversely, it was observed that fresh isolates, obtained from
5 Aeromonadaceae Representative (Aeromonas salmonicida)
The A-Layer
The A-layer is now thought to be the product of a single chromosomal gene (Belland
and Trust 1985 ), is produced in vivo (Ellis et al. 1997 ) and contributes to survival in
macrophages (Daly et al. 1996 ). The virulence array protein gene A ( vapA ) , which
encodes the A-protein has been sequenced, and differences noted in the amino acids
between typical and atypical isolates, with homogeneity among the former but heterogeneity with the latter. These differences undoubtedly lead to antigenic differences among atypical isolates (Lund and Mikkelsen 2004 ). First reported by Udey
and Fryer ( 1978 ), and resulting from detailed electron microscopic studies, the
A-layer was determined to be correlated with virulence (e.g. Madetoja et al. 2003 );
an insulin-binding capacity for the A-layer has been documented (Nisr et al. 2012 ).
It was observed that virulent strains possessed the A-layer, whereas avirulent isolates did not. In addition, the presence of the A-layer was found to correspond with
strong auto-agglutinating properties of the organism, and to the adhesion to fi sh
tissue culture cells. The auto-agglutination trait has been found to be infl uenced by
temperature, with weak and strong auto-agglutination at 25 and 15–20 °C, respectively (Moki et al. 1995 ). The presence of the A-layer may confer protection against
phagocytosis and thus destruction by macrophages (Olivier et al. 1986 ; Graham
et al. 1988 ). Essentially, these workers noted that avirulent cells, i.e. those without
an A-layer, were phagocytosed and destroyed when virulent cells with A-layer were
more resistant. Moreover, the bacteriocidal activity of macrophages was stimulated
by prior exposure to low doses of Ren. salmoninarum, but inhibited by high amounts
of living or dead renibacterial cells or the p57 antigen (Siegel and Congleton 1997 ).
Interestingly, it was deduced that living and formalised virulent cells, in the absence
of serum, attracted macrophages more readily than avirulent cells after a period of
90 min (Weeks-Perkins and Ellis 1995 ). The surface layer may inhibit growth at
30 °C, enhance cell fi lamentation at 37 °C, and enhance uptake of the hydrophobic
antibiotics streptonigrin and chloramphenicol (Garduño et al. 1994 ). Following the
intravenous injection of purifi ed A-layer protein into Atlantic salmon, the protein
located to the epithelial cells in renal proximal tubules of the head kidney (Stensvåg
et al. 1999 ).
For its formation, Belland and Trust ( 1985 ) reasoned that the A-layer subunits
pass though the periplasm and across the outer membrane for assembly on the cell
surface. A requirement for the presence of O-polysaccharide chains, for which the
AbcA protein is involved in biosynthesis (Noonan and Trust 1995 ) on the LPS was
reported as necessary for the assembly of A-layer (Dooley et al. 1989 ). These virulent, auto-agglutinating forms produce characteristic deep blue colonies on CBB
agar (Wilson and Horne 1986 ; Bernoth 1990 ). Sakai ( 1986a , b ) postulated that a
possible mechanism for auto-agglutination and adhesion could be attributed to the
presence of net negative electrical charge in the interiors or on the surfaces of cells.
In particular, pathogenic cultures were highly adhesive (Sakai 1987 ). It should be
emphasised that Udey and Fryer ( 1978 ) determined that strains maintained for long
periods in laboratory conditions were not auto-agglutinating, and demonstrated
reduced virulence. Conversely, it was observed that fresh isolates, obtained from
5 Aeromonadaceae Representative (Aeromonas salmonicida)