534
intradermal injection. Much larger inocula resulted in sizeable mortalities. For
example, Evelyn ( 1971 ) determined that Oncorhynchus keta and O. nerka died
within 48 h of receiving, by i.p. injection, 0.1 ml containing 10
7 viable cells of V.
anguillarum. In a much more spectacular demonstration of virulence, Sawyer et al.
( 1979 ) established 80–100 % mortality in a population of Atlantic salmon following
exposure to 1–2.5 × 10
5 cells/ml as a bath for 1 h. In this demonstration, the fi sh
were maintained at a water temperature of 10–15 °C. However, temperature shocking does exacerbate mortality. Thus in one series of experiments using rainbow
trout, the temperature was decreased from 23 to10°C resulting in a signifi cantly
increased level of mortality; an increase which was not correlated with an impairment in immune parameters (Aoshima et al. 2005 ).
Turbot larvae have been successfully challenged with V. anguillarum orally via
live feed (Grisez et al. 1996 ; Planas et al. 2005 ). Using 10
3 Artemia nauplii/ml and
10
9 V. anguillarum cells/ml, the recipient fi sh died within 4 days (Grisez et al. 1996 ).
Similarly, feeding with rotifers containing V. anguillarum cells led to a successful
infection of turbot larvae (Planas et al. 2005 ).
The precise nature of the virulence mechanism of V. anguillarum has prompted
some excellent work. With the advent of random genome sequencing, a strain,
H775-3, was examined, and 40 genes, which may well be related to virulence identifi ed, of which 36 genes were considered to be novel to V. anguillarum, and included
genes for capsule biosynthesis, enterobactin, haemolysin, fl agella, LPS biosynthesis, pilus and protease (Rodkhum et al. 2006 ). Rainbow trout skin epithelial cells
were shown to phagocytize the pathogen. Moreover, use of mutants has shown that
V. anguillarum requires the LPS O-antigen to evade phagocytosis. By adding mannose to epithelial cells, phagocytosis was inhibited, which indicates that a mannose
receptor is involved with uptake of the pathogen. Interestingly, O-antigen transport
mutants did colonize skin but did so the intestines of rainbow trout (Lindell et al.
2012 ). The highlight of the early studies was the discovery that bona fi de strains of
serogroup O1 of the pathogen contained a virulence plasmid, which was associated
with an iron-uptake system expressed under iron-limited conditions (Crosa et al.
1977 ; Crosa 1980 ; Crosa 1980 ; Wolf and Crosa 1986 ; Chen et al. 1996 ). This plasmid, designated pJM1 and of 67 kb, has been fully sequenced (Di Lorenzo et al.
2003 ), and is always present in virulent isolates (Pedersen et al. 1996a , b , 1997b )
and may be included on a transposon-like structure (Tolmasky and Crosa 1995 ), but
absent from those of low virulence. The genes involved in the biosynthesis of anguibactin are located on the plasmid and chromosome. However, the genes for the outer
membrane receptor, FatA, are located only on the plasmid (López et al. 2007 ).
Interestingly, the pJM1 plasmid has been found in some avirulent isolates (Pedersen
et al. 1997b ). Yet, virulence may be attenuated by curing this plasmid (Crosa 1980 )
or by deleting three plasmid encoded gene products (Singer et al. 1991 ). The role for
pJM1 concerns specifying an iron-sequestering mechanism, i.e. the low molecular
weight siderophore anguibactin for which the precursor is chromosome-mediated
2,3-dihydroxybenzoic acid (Chen et al. 1994 ), and specifi c iron-transport proteins,
of which the angR protein (this is regulated by the regulatory gene angR [Salinas
and Crosa 1995 ], which has been reported as similar to bacteriophage P22; Farrell
10 Vibrios
intradermal injection. Much larger inocula resulted in sizeable mortalities. For
example, Evelyn ( 1971 ) determined that Oncorhynchus keta and O. nerka died
within 48 h of receiving, by i.p. injection, 0.1 ml containing 10
7 viable cells of V.
anguillarum. In a much more spectacular demonstration of virulence, Sawyer et al.
( 1979 ) established 80–100 % mortality in a population of Atlantic salmon following
exposure to 1–2.5 × 10
5 cells/ml as a bath for 1 h. In this demonstration, the fi sh
were maintained at a water temperature of 10–15 °C. However, temperature shocking does exacerbate mortality. Thus in one series of experiments using rainbow
trout, the temperature was decreased from 23 to10°C resulting in a signifi cantly
increased level of mortality; an increase which was not correlated with an impairment in immune parameters (Aoshima et al. 2005 ).
Turbot larvae have been successfully challenged with V. anguillarum orally via
live feed (Grisez et al. 1996 ; Planas et al. 2005 ). Using 10
3 Artemia nauplii/ml and
10
9 V. anguillarum cells/ml, the recipient fi sh died within 4 days (Grisez et al. 1996 ).
Similarly, feeding with rotifers containing V. anguillarum cells led to a successful
infection of turbot larvae (Planas et al. 2005 ).
The precise nature of the virulence mechanism of V. anguillarum has prompted
some excellent work. With the advent of random genome sequencing, a strain,
H775-3, was examined, and 40 genes, which may well be related to virulence identifi ed, of which 36 genes were considered to be novel to V. anguillarum, and included
genes for capsule biosynthesis, enterobactin, haemolysin, fl agella, LPS biosynthesis, pilus and protease (Rodkhum et al. 2006 ). Rainbow trout skin epithelial cells
were shown to phagocytize the pathogen. Moreover, use of mutants has shown that
V. anguillarum requires the LPS O-antigen to evade phagocytosis. By adding mannose to epithelial cells, phagocytosis was inhibited, which indicates that a mannose
receptor is involved with uptake of the pathogen. Interestingly, O-antigen transport
mutants did colonize skin but did so the intestines of rainbow trout (Lindell et al.
2012 ). The highlight of the early studies was the discovery that bona fi de strains of
serogroup O1 of the pathogen contained a virulence plasmid, which was associated
with an iron-uptake system expressed under iron-limited conditions (Crosa et al.
1977 ; Crosa 1980 ; Crosa 1980 ; Wolf and Crosa 1986 ; Chen et al. 1996 ). This plasmid, designated pJM1 and of 67 kb, has been fully sequenced (Di Lorenzo et al.
2003 ), and is always present in virulent isolates (Pedersen et al. 1996a , b , 1997b )
and may be included on a transposon-like structure (Tolmasky and Crosa 1995 ), but
absent from those of low virulence. The genes involved in the biosynthesis of anguibactin are located on the plasmid and chromosome. However, the genes for the outer
membrane receptor, FatA, are located only on the plasmid (López et al. 2007 ).
Interestingly, the pJM1 plasmid has been found in some avirulent isolates (Pedersen
et al. 1997b ). Yet, virulence may be attenuated by curing this plasmid (Crosa 1980 )
or by deleting three plasmid encoded gene products (Singer et al. 1991 ). The role for
pJM1 concerns specifying an iron-sequestering mechanism, i.e. the low molecular
weight siderophore anguibactin for which the precursor is chromosome-mediated
2,3-dihydroxybenzoic acid (Chen et al. 1994 ), and specifi c iron-transport proteins,
of which the angR protein (this is regulated by the regulatory gene angR [Salinas
and Crosa 1995 ], which has been reported as similar to bacteriophage P22; Farrell
10 Vibrios