The aquaculture industry has developed significantly over recent decades and is, today, one of the fastest-growing food production sectors in the world. One of the most important problems that affect aquaculture is the appearance of infectious diseases. Among bacterial diseases affecting cultured salmonid fish, Bacterial Cold-Water Disease, caused by Flavobacterium psychrophilum, produces high mortality and morbidity and consequently, economical losses worldwide.

1. T
he aquaculture industry has
developed significantly over recent
decades and is, today, one of the
fastest-growing food production
sectors in the world. One of the most
important problems that affect aquaculture
is the appearance of infectious diseases.
Among bacterial diseases affecting cultured
salmonid fish, Bacterial Cold-Water Disease,
caused by Flavobacterium psychrophilum,
produces high mortality and morbidity and
consequently, economical losses worldwide.
Attempts to control
this situation through
the use of antimicrobials
have been limited due to
the rapid appearance of
antibiotic-resistant bacteria.
Moreover, the determi-
nants of antibiotic resist-
ance that have emerged
and selected in this aquatic
environment have the
potential of being trans-
mitted by horizontal gene
transfer to bacteria of the
terrestrial environment,
including human and animal
pathogens.
Evidence indicating that
antibiotic-resistant bacteria
and antibiotic resistance
determinants pass from the
aquatic to the terrestrial environment has
resulted in a drastic restriction of the use of
antibiotics in aquaculture in many countries. For
this reason, it is important to find new products
with antimicrobial activity without the afore-
mentioned undesirable effects of antibiotics.
In this sense, natural food additives or phy-
tobiotics, which combined different mecha-
nisms of action against pathogenic bacterial
species (bactericidal/ bacteriostatic activities,
Quorum Sensing inhibition), are potential
candidates for the development of prevention
strategies in aquaculture. This work describes
the effect of the dietary administration to
rainbow trout of the phytobiotic Liptofry
(Liptosa S.A, Spain) in the improvement
of disease resistance against Flavobacterium
psychrophilum.
In this study 4000 healthy rainbow trout,
distributed in two experimental groups (2000
individuals/each) were maintained in an aqua-
culture facility of the north of Spain, until
its transfer to the aquarium of the Faculty
of Biology of the University of Santiago de
Compostela. Rate of food supply of both
groups was established based on the require-
ments of the fish, taking as
a reference the parameters
of size and weight of the
specimens and the tem-
perature of the water.
• Group 1: Fish fed with
feed supplemented with
the phytobiotic Liptofry
manufactured by Lípidos
Toledo SA (Liptosa SA,
Spain).
• Group 2: Fish fed with
no supplemented feed
(control fish).
The usefulness of
feeding the fish with diet
supplemented with phyto-
biotics in the prevention
of Bacterial cold-water dis-
ease (BCWD) in rainbow
trout was determined by
Nutraceutics for the control of
bacterial coldwater disease
by C. Fernández-Álvarez, Facultad de Biología (CIBUS), Y. Torres-Corral, Facultad de Biología (CIBUS), G. Coscelli, Facultad de
Ciencias Veterinarias, Universidad Nacional de Rosario, Argentina, A. R. Sánchez-Arévalo, Lípidos Toledo S.A; Madrid, Spain,
A. Martínez, Lípidos Toledo S.A; Madrid, Spain, M.I. Quiroga, Facultad de Veterinaria, Universidad de Santiago de Compostela,
Spain, Y. Santos, Facultad de Biología (CIBUS)
14 | INTERNATIONAL AQUAFEED | November-December 2015
FEATURE

2. experimental infection, using a virulent strain
RBT4.1.04 of the species F. psychrophilum.
With this aim, 120 fish of each experimental
group (group 1 and group 2) were anaesthe-
tised by immersion in tricaine methane sul-
fonate (MS-222, Sigma) (60mg/L) and infected
by intra-peritoneal injection of a bacterial sus-
pension containing 109 CFU/mL. Differences
in survival between fish fed with phytobiotics
(Group 1) and control fish (Group 2) after
challenge with Flavobacterium psychrophilum
were analysed by Chi-square test (P<0.05)
(Figure 1).
Histopathological analy-
sis was performed in order
to evaluate the effect of the
administration of the phyto-
biotic Liptofry in fish tissues.
Tissue samples from control and
Liptofry-feeded fish were taken
at 24, 48, 72 y 96 h after experi-
mental infection. Bouin’s fixed
samples were embedded in par-
affin wax, sectioned at 3 μm in
thickness, and stained with hematoxylin-eosin
(H-E), Phosphotungstic acid-haematoxylin
stain (PTAH) and Periodic acid–Schiff stain
(PAS) for light microscopy observation.
In our study, fish fed with the Liptofry sup-
plemented diet (group 1) showed an average
cumulative mortality of 27 following challenge
experiment, compared to 62 percent mortal-
ity observed in control fish (group 2) (Table
1). The survival of fish fed with the phytobiotic
Liptofry was significantly higher than those of
the control group (p<0.05).
Histopathological analysis showed that fish
of both experimental groups showed similar
lesions, characterised by a moderate inflam-
matory response at the coelomic cavity,
necrotic areas affecting the spleen, liver, and
pancreatic lobes, and degenerative lesions of
renal tubules, showing pyknosis and dequa-
mation of epitelial cells (Figure 2),
Results of this study, suggest that the use
of feed supplemented with the phytobiotic
Liptofry in the diet of rainbow trout may con-
tribute to the prevention of diseases caused
by the bacterial pathogen Flavobacterium
psychrophilum. Thus, these diet supplements
could be used as an alternative to antibiotics
or vaccines in the control and/or prevention of
infectious diseases. Although distribution of the
inflammatory response in fish of groups 1 and
2 infected with Flavobacterium psychrophilum
was similar, the severity and extent of lesions,
and the number of animals affected were
higher in the control group. These findings
might correlate with significant
differences of mortality in groups
one and two.
Acknowledgements
This work was supported by
Grant CDTI-IDI-20140147 from
the Centro para el Desarrollo
Tecnológico e Industrial (CDTI),
Ministerio de Economía y
Competitividad, Spain
Table 1. Experimental infection with F. psychrophilum to evaluate the
effect of the Liptofry supplemented diet in the prevention of BCWD
Experimental group
Inoculated dose
(CFU/fish)
Nº death fish/
Nº inoculated fish
% Mortality
Group 1
Fish treated with the
phytobiotic Lyptofry
4.2 x 108 32/120 27
Group 2
Control fish
4.2 x 108 74/120 62
November-December 2015 | INTERNATIONAL AQUAFEED | 15
FEATURE
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