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FEATURE

Immunostimulation
in aquatic animals
by Philippe Tacon, global aquaculture manager, Phileo

survey made at the end of an aqua industry forum


meeting in Vietnam last year has shown that for 63
percent of the participants, the most limiting challenge
for developing aquaculture was health and disease
management. Indeed, in recent years, we have seen numerous
diseases appearing and impacting aquaculture production, such
as WSSV and EMS in shrimp, or Infectious Salmon Anemia
(ISA) in salmonids. Working around the classic Host-PathogenEnvironment triad, new technologies and management techniques
have been developed to better control diseases in aquatic
animals: vaccination, which has led to the decrease of antibiotic
use in salmonids; biosecurity procedures in hatcheries and in
farms; biofloc technology. All of these technologies have proven
successful. Their further development and expanded use will
certainly improve the way aquatic animals are farmed.
Another strategy is to increase the health of the animal through
feeding, and this magazine might be a good place to discuss it. Well
balanced diets can certainly improve the health status of a fish or a
shrimp, but in some challenging conditions, like a pathogen infection,
the use of immune stimulants can be required to enhance the
response of the immune system.
When studying immune stimulation, it is important to understand
that the immune system of aquatic animals differs not only between
theirs and the mammalian one but also between teleost and
crustacean. Fish are the first group in which a specific immune
system appears in the evolutionary tree. The fish immune system
therefore has a greatly inferior performance to that of mammals
(see Tort et al 2003). It is less specific, less sensitive and has only
oneclass of antibodies (IgM). Fish being poikilothermic animals, it is
highly dependent on temperature, low temperature slowing down
the immune response up to 10 to 12 weeks. Fish rely by then more
on their non-specific immune system (also called innate immunity) to
fight against pathogens. The innate immune system recognises nonself molecules that could be of foreign origin - also called pathogen
associated molecular patterns (PAMP) - and molecular patterns
exposed though damage to the host. These patterns are recognised
by germline-encoded pattern recognition receptors (PRR) or pattern

recognition proteins (PRP). These molecular patterns can be for


example peptidoglycans and lipopolysaccharides from bacteria cell
walls, fungal b1, 3-glucan, viral double-stranded RNA and bacterial
DNA (see Magnadottir 2006 for an overview of fish innate immunity).
Fish innate immunity starts with first barrier defences such as mucus;
it traps pathogens and includes lysozymes, antibacterial peptides
which can eliminate pathogens. Neutrophils and macrophages are
key cells of the innate immune complex as they can phagocytose
pathogens (a mechanism which is not temperature dependent) and
release Reactive Oxygen species, which are toxic to pathogens.
Completing this cellular response, the humoral response implicates
the synthesis and release of antimicrobial components.
In shrimp, where the picture is even simpler as they rely only on
innate immunity, we find the same type of mechanisms in place as
in fish with phagocytosis performed by granulocytes (a specific form
of the blood hemocyte cells) and humoral response. However the
most effective mechanism of invertebrates (as arthropods) is cellular
melanotic encapsulation. This requires the combination of circulating
hemocytes and several associated proteins of the prophenoloxidase
(proPO) activating system. Recognition of PAMPs such as LPS and
-1, 3 glucans by PRPs is an essential step for the activation of the
proPO cascade (Amparyup et al 2013).
Stimulation of the innate immune system, which would enhance
the speed and the effect of the immune response, is therefore
possible by mimicking the effect of PAMP on PRR and PRP. In
that regard, beta glucans have been studied for a long time in
aquaculture and seem the ideal immune stimulant in aquaculture
(see Meena et al 2013 and Ringo et al 2012) as they can specifically
activate macrophages in fish and the proPO cascade in shrimp.
Parietal fractions, such as Safmannan are extracted from
a selected Saccharomyces cerevisiae strain respecting strict EU
manufacturing control standards. They contain beta glucans,
mannan oligosaccharides that are all activators of the immune
system (Song et al 2014).
Earlier internal trials have shown that yeast cell walls and parietal
fractions have different effects in mycotoxin binding and immunity
in aquatic animals. Indeed several trials done at the Hellenic Center
for Marine Research in Greece have shown that yeast fraction

18 | INTERNATIONAL AQUAFEED | March-April 2015

FEATURE
products with similar manna/
glucan ratios from Phileo, Lesaffre
Table 1: Formulation and compositions of experimental diets (%).
Animal Care Business Unit, have
Ingredients
very different effects in the
stimulation of immune parameters
Fishmeal 38.5
25
25
25
25
25
and in survival following challenge
Soybean protein concentrate
20
20
20
20
20
20
in Vibrio anguillarum. It looks like
Soybean meal
0
21
21
21
21
21
not only the mannan and glucan
Wheat flour
21
21
21
21
21
21
content is of importance, but the
strain and the drying processes
Fish oil
6
6.4
6.4
6.4
6.4
6.4
are also key parameters to ensure
Monocalcium
1
2.1
2.1
2.1
2.1
2.1
a good effect in aquatic animals.
phosphate(Ca(H2PO4)2)
Another concept that came out
Microcrystalline cellulose 10.1
1
0.975
0.95
0.9
0.8
of these trials was that there is a
Phospholipid (93%)
2
2
2
2
2
2
threshold of yeast material to be
Choline chloride(50%) 0.4
0.4
0.4
0.4
0.4
0.4
ingested before it starts to kick in
and improve the immune system.
Vitamin and mineral Premixa
1
1
1
1
1
1
Product origin, quality, dosages
Methionine hydroxy analog-Ca(98%)
0
0.1
0.1
0.1
0.1
0.1
and duration of treatment are all
Safmannan (mg/kg)
0
0
250
500
1000
2000
clearly linked.
Analyzed chemical compositions(dry matter basis %)
A trial has been undertaken to
further study a dose response of
Crude protein 47.6
48.2
48.7
47.9
48.4
48.5
Safmannan in a marine species.
Crude lipid 12.1
12.0
11.7
11.6
11.8
11.6
The objective was twofold:
Crude ash 7.86
8.51
8.46
8.52
8.59
8.41
investigate the influence of
parietal fractions in diets with a
Gross energy(MJ/kg) 21.5
21.3
21.4
21.5
21.4
21.6
reduced amount of fishmeal, and
determine the dosage needed for
diets were supplemented with 0 (SBM), 250, 500, 1000 and 2000
an optimum immune response (Yu et al 2014).
Six diets were designed (see table 1): a high fishmeal diet with g/T of Safmannan. Juvenile Japanese seabass (18 g) were selected
38.5 percent fish meal inclusion and no soybean meal (HFM) and 5 and distributed into 280 L tanks after 24 h starvation with 30 fish
diets with 25 percent fishmeal and 20 percent soybean meal. These per tank, and six tanks per treatment. The water temperature was

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March-April 2015 | INTERNATIONAL AQUAFEED | 19

FEATURE

maintained. Fish were fed to apparent satiation twice daily at 08:00


and 15:00 for 72 days.
At the end of the treatment period fish were anesthetsed, weighed
and viscera and blood were sampled. Intestine samples from the FM,
Y0, Y4 and Y5 groups were removed from 2 fish in each replicate tank
at the end of trial (12 fish per treatment) and processed for histology
analysis (H & E staining). Morphological parameters associated with
SBM-induced enteritis of anterior and distal intestines, including the
height of mucosal folds (HMF), width of mucosal folds, lamina propria
and connective tissue were quantified.
After all samples were taken, 40 fish of each treatment (67 fish
per tank) were divided into 2 groups and transferred into a still water
system with temperature at 26 1 C. The fish were fed as before and
recovered from weighing and sampling stress by a 2-week acclimation.
Then they were challenged by intramuscular injection with Aeromonas
veronii (CGMCC No. 4274) at 8 104 cells/100 g body weight. Ten
fish from each tank were sampled for plasma immune parameters
two days after challenge and the others (20 fish per treatment) were
recorded for 7-day cumulative survival rate without any food.
This study showed a lower growth of SBM diets as expected
compared to HFM diets, but an even lower growth with the 500g/T
treatment, and a much better growth at 2000 g/T (Fig1). These results
can be correlated to a wider width of mucosal folds in anterior and
distal intestinal in SBM diets compared to HFM diets suggesting a
negative effect of these diet on intestinal health, and also to a higher
height of mucosal folds in the 2000 g/T group (Fig1). This suggests that
Safmannan at 2000 g/T was able to compensate the negative effect
of soybean meal and increase gut health leading to a better growth.
The study also shows that IgM levels were significantly elevated
after the bacterial challenge in the diet containing parietal fractions
at 500g/T (Fig2) indicating a strong immune stimulation. The levels
decrease as the yeast parietal fraction concentration is increased
showing a potential fatigue of the immune system. This is confirmed
by the survival of the fish after the challenge. The optimum dosage
was 500g/T of Safmannan, whereas higher dosage did not improve
survival. Remarkably, we can see this optimum dosage for immune
stimulation was also the one giving the lowest growth, confirming
hypothesis that the strong stimulation of the immune system is at the
expense of the growth potential of the fish.
This study highlights the duality of role of parietal fractions in fish
depending on the dosage and feed composition: they can be used
either as gut health enhancer (high dosage) or immune enhancer (low
dosage).
Formulators and farmers can benefit from using this efficient
and sustainable solution against pathogens but they need to choose
quality products and work with proper (and proven) dosages and
administration durations.

Figure 1 growth and intestinal health parameters in


Japanese seabass following a treatment with yeast parietal
fractions. Values with different subscripts are significantly
different (P<0.05) (Yu et al 2014)

Figure 2 immune parameters and cumulative mortality


following treatment with parietal fractions.
Values with different subscripts are significantly different
(P<0.05) between treatments, asterisks show a different
between time. For the bacterial challenge, Safmannan 500 is
different than control at P<0.05 (Yu et al 2014)

20 | INTERNATIONAL AQUAFEED | March-April 2015

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