429
Laboratory-based studies using sterilised fresh water have shown that Fla. psychrophilum has the capacity to survive for 300 days at 15 °C; in 30 % sodium chloride culturability was <1 day (Madetoja et al. 2003). However in the absence of a
normal microfl ora, the relevance of the data to explaining survival of the pathogen
in the natural environment is debatable. Survival studies revealed that the organism
stopped multiplying but the numbers remained fairly constant in stream water for 16
weeks, and then declined. The cells became smaller and more rounded. Culturable
cells could not be recovered on Cytophaga agar after 19 weeks, but resuscitation in
cytophaga broth was possible for up to 36 weeks. In distilled water, culturability
was lost after 1 h (Vatsos et al. 2003 ). The pathogen has been detected on algae
growing on the surface of stones in rivers that contain diseased (with Fla. psychrophilum) fi sh (Amita et al. 2000 ).
Colonisation of fi sh may be a forerunner for the development of disease. For
example, colonisation of eyed rainbow trout ova with Fla. psychrophilum has been
considered to lead to the development of RTFS (Rangdale et al. 1997b ). Using a
modifi ed Anacker and Ordal’s medium, the pathogen was recovered from ovarian
fl uid of 2/15 hen fi sh, egg surfaces 14 days after fertilisation, but not from milt
(Rangdale et al. 1996 ).
Ribotyping and plasmid profi ling appear to be useful for epizootiology. In particular, several ribotypes have been recognised among 85 isolates, and determined
to be associated with the fi sh species from which the cultures were obtained
(Chakroun et al. 1998 ).
There is no dispute that these organisms are capable of causing severe losses to
fi sh populations. Coldwater disease may cause losses of up to 50 % of the fi sh population, as determined for coho salmon fry (Rucker et al. 1953 ). This disease is especially troublesome at low water temperatures (Holt et al. 1989 ), i.e. ≤15 °C.
Pathogenicity
Fla. psychrophilum has been shown to attach to and even colonise the surface of fi sh
(rainbow trout) eggs (Vatsos et al. 2001 , 2006 ), and is certainly capable of inducing
infection of eggs (Atlantic salmon) (Cipriano 2005 ). Exposure of disinfected eyed
ova of rainbow trout with an extremely high dose of 10
10 cells of Fla. psychrophilum /ml in PBS for 30 and 60 min at 10 °C led to the development of clinical signs
of RTFS (Rangdale et al. 1997b ). Rainbow trout unfertilized eggs were immersed in
lesser doses of 10
7 CFU/ml and above, leading to the presence of the pathogen in the
eggs. Furthermore, when mature hen fi sh were injected i.p. with 3.3 × 10
9 CFU/fi sh
some 5–9 days before ovulation, the resulting intra-ovum infection revealed heavy
contamination in the ovarian fl uid, i.e. >10
6 CFU/ml. The conclusion was reached
that the pathogen could contaminate eggs in ovarian fl uid becoming internalized
during water hardening (Kumagai and Nawata 2010 ). Nanoinjection of newly fertilized rainbow trout eggs with 10
1 – 10
3 CFU/egg led to higher mortalities (95–100 %
mortalities of the eggs) than the uninfected controls (0 % mortality) at 70 days
Flavobacterium psychrophilum
Laboratory-based studies using sterilised fresh water have shown that Fla. psychrophilum has the capacity to survive for 300 days at 15 °C; in 30 % sodium chloride culturability was <1 day (Madetoja et al. 2003). However in the absence of a
normal microfl ora, the relevance of the data to explaining survival of the pathogen
in the natural environment is debatable. Survival studies revealed that the organism
stopped multiplying but the numbers remained fairly constant in stream water for 16
weeks, and then declined. The cells became smaller and more rounded. Culturable
cells could not be recovered on Cytophaga agar after 19 weeks, but resuscitation in
cytophaga broth was possible for up to 36 weeks. In distilled water, culturability
was lost after 1 h (Vatsos et al. 2003 ). The pathogen has been detected on algae
growing on the surface of stones in rivers that contain diseased (with Fla. psychrophilum) fi sh (Amita et al. 2000 ).
Colonisation of fi sh may be a forerunner for the development of disease. For
example, colonisation of eyed rainbow trout ova with Fla. psychrophilum has been
considered to lead to the development of RTFS (Rangdale et al. 1997b ). Using a
modifi ed Anacker and Ordal’s medium, the pathogen was recovered from ovarian
fl uid of 2/15 hen fi sh, egg surfaces 14 days after fertilisation, but not from milt
(Rangdale et al. 1996 ).
Ribotyping and plasmid profi ling appear to be useful for epizootiology. In particular, several ribotypes have been recognised among 85 isolates, and determined
to be associated with the fi sh species from which the cultures were obtained
(Chakroun et al. 1998 ).
There is no dispute that these organisms are capable of causing severe losses to
fi sh populations. Coldwater disease may cause losses of up to 50 % of the fi sh population, as determined for coho salmon fry (Rucker et al. 1953 ). This disease is especially troublesome at low water temperatures (Holt et al. 1989 ), i.e. ≤15 °C.
Pathogenicity
Fla. psychrophilum has been shown to attach to and even colonise the surface of fi sh
(rainbow trout) eggs (Vatsos et al. 2001 , 2006 ), and is certainly capable of inducing
infection of eggs (Atlantic salmon) (Cipriano 2005 ). Exposure of disinfected eyed
ova of rainbow trout with an extremely high dose of 10
10 cells of Fla. psychrophilum /ml in PBS for 30 and 60 min at 10 °C led to the development of clinical signs
of RTFS (Rangdale et al. 1997b ). Rainbow trout unfertilized eggs were immersed in
lesser doses of 10
7 CFU/ml and above, leading to the presence of the pathogen in the
eggs. Furthermore, when mature hen fi sh were injected i.p. with 3.3 × 10
9 CFU/fi sh
some 5–9 days before ovulation, the resulting intra-ovum infection revealed heavy
contamination in the ovarian fl uid, i.e. >10
6 CFU/ml. The conclusion was reached
that the pathogen could contaminate eggs in ovarian fl uid becoming internalized
during water hardening (Kumagai and Nawata 2010 ). Nanoinjection of newly fertilized rainbow trout eggs with 10
1 – 10
3 CFU/egg led to higher mortalities (95–100 %
mortalities of the eggs) than the uninfected controls (0 % mortality) at 70 days
Flavobacterium psychrophilum