Effect of an immuno-modulating algal extract on disease prevention in aquaculture Maarten Jay van Schoonhoven, Aqua Care Manager Olmix [email protected]
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Effect of an immuno-modulating algal extract on disease prevention in
aquacultureMaarten Jay van Schoonhoven, Aqua Care Manager Olmix
Why Seaweeds:
Natural
Bioactive
Renewable
Blue biotechnology from the sea
Common seaweedsFSC guide
Phaeophyta (Brown Seaweeds)• 1700 species • Prodominantly brown due to carotenoid fucoxanthin• Primary polysaccharides: alginates, laminarins, fucoidans and
cellulose
Algae Seaweed Classification
4
Chlorophyta (Green Seaweed) • 1500 species• Dominated by chlorophyll a and b• Primary polysaccharide are the Ulvans
Rhodophyta (Red Seaweed) • 6100 species• Pigments Phycoerythrim and phycocyanin• Primary ploysaccharides are agars and carrageenans
Seaweeds are made up of :- Carbohydrates (20-70% dry weight)
- mainly polysaccharides :- Alginates (brown algae)- Carraghenanes (red algae)- Ulvans (green algae)
- Proteins (1-35% dry weight)- Lipids (0,5-4% dry weight)
Minerals (10-30% dry weight)- Iron, Iodine, Copper, Potassium, Sodium, Calcium,- Magnesium, Sulfur, Phosphorous, Chlorine, Manganese…- Many other trace elements.
Vitamins - A, B1, B2, B6, B12, C, D3, E, K
PigmentsPhycoerythrin (antioxidant)Carotenoids
Marine SulfatedPolysaccharides
MSP Chemically and biologically activesubstances used in biochemistry andmedicine.
Unique chemical compositions
With different structuralcompositions depending on theirorigin (brown, green, red…)
The new focus on sulfated polysaccharides and their bio-pharmacological activities is due to their immuno-modulatory, anti-hyperlipidemic, antiviral, antibacterial and anti-tumor effects.
SULPHATED POLYSACCHARIDES
Unbranched homo-polysaccharide
Branched homo-polysaccharide
Branched hetero-polysaccharide
Branched sulfated hetero-polysaccharide
Sugar unit
Sulfate
SS
S
S
S S
Branched sulfatedhetero-polysaccharide
Sugar unit
Sulfate
Linear polysaccharide
Branched hetero-polysaccharide
Homo-polysaccharideramifié
1° - Diversity of sugar units (e.g: glucose, fructose)2° - Presence of rare sugars (e.g: rhamnose, fucose)3° - Branching Increased biological activities4° - Sulfation Unique biological activities
All these parameters show a phylogenetic similarity with the polysaccharides of the animal kingdom which explains the unique biological activities.
S
SS
S
S
S S
HEPARIN• Anti-inflammatory• Immunomodulation• Antitumoral• Anticoagulant and antitrombotic
MSP• Anti-inflammatory (Kim et al. 2009; Hong et al. 2011)• Immunomodulation (Kim et al. 2012; Berri et al. 2013)• Mucin production stimulation (Barcelo et al. 2000)• Antioxidant (Baky et al. 2009; Zhang et al. 2010; Souza et
al. 2006)• Antilipidemic (Hassan et al. 2011)• Antiviral (Wang et al.2012)• Antitumoral (Veeraperumal et al. 2012; Baky et al. 2009)• Anticoagulant and antitrombotic
Algal active ingredients properties:
• Biological activitiesMarine Algae Sulfated polysaccharides (MSP)
- 3D structure- Rare sugars- Sulfate content- Phylogenetic analogy with
animal glycosaminoglycanes
• Biostimulation
• Immunomodulating• Anti-hyperlipidemic• Mucin secretion• Antioxidant• Antitumor• Antiviral• Antistress• …
Can serve the whole food chain
TEST 1. Intestinal Porcine Epithelial Cells (IPEC-1) ‘s differentiation and stimulation of the production of « Messenger molecules » of the immune system
• 0,25x105 IPEC-1 cells/cm2
• 3 days of cell culture, until the observation of confluence
Cell culture : 10 to 14 days withDexamethasone (10-7M)
Analyze of mediator gene expression by RT-qPCR
Trans epithelial resistance, immunohistochemistry, and microscopic observations
Addition of the MSP at different concentrations
CYTOKINES AND CHEMOKINES’S EXPRESSION IN IPEC-1 EPITHELIAL CELLS
Increased expression of several « Messenger molecules » of the immune system in differentiated
IPEC-1 cells when they are stimulated with MSP at the concentration of 1mg/ml. (**: P<0.01)
Expr
essio
n re
lativ
e
CCL20Lymphocytes and dendritic
cell’s recruitment
DMEM 1 0.1 0.01 LPS0
2000
4000
6000
8,1
**
Dose (mg/ml)
IL-8Neutrophils'
recruitment & phagocytosis
Dose (mg/ml)
0
100
200
11,4
1 0.1 0.01 LPS
**
DMEMDose (mg/ml)
IL-1αB Lymphocytes:
proliferation and differentiation.
1 0.1 0.01 LPS0
10
20
30
**
6,2
DMEM
TNFαPhagocytosis &
neutrophils’ chemiotaxis
Dose (mg/ml)1 0.1 0.01 LPS
0
25
50
75
100
**
6,2
DMEM
RESULTS
Hayashi et al., 2003 ; Makarenkova, I.,2012
Receptors(TLR, MR, LR…)
Epithelial cell
3. Cell function activationProduction of :
- Cytokines (e.g: TNFα, IL-1, IL-6…)- Chemokines (e.g: CCL2, CCL20…)
2. Receptor activation
4. Leucocyte activation NK, macrophages …= Innate ImmunityLymphocytes T , B…= Adaptive immunity
MSP 1. MSP binding on the receptors
Destruction of the microbebefore a strong proliferation
Transcription
Signal Transduction
Nucleus
MSP Immunomodulating
Use of Searup and Digestsea in freshwater fish production in Poland
15
Common diseases in Polish Carp production
16
Aims for Polish Carp
- KHV (Koi Herpes Virus)- Reduce antibiotic use and increase natural defense against infectious skin
disease (Carp Erythrodermatitis)- Decrease occurrence of tape worm in digestive tract
Common diseases in Polish Carp production
17
• KHV is a viral disease affecting gills, kidney and skin• Can lead up to 95% mortality• Often triggered by temperature fluctuations spring and fall
• Carp Erythrodematitis is caused by Aeromonas normally present in the pond environment
• Disease is triggered by stress which lower immune defense
• Tapeworm (Khawia sinensis, Caryophlaeus laticeps and Bothriocephalusageilognathi) most common in summer
• Few symptoms, other than reduced feed intake and poor growth
Field trials on Polish carp
18
• Application• 1ml Searup/10kg fish biomass• 1ml Digestsea/10kg fish biomass
• 3x/week initially (carp are fed every 2nd day)• Then 1x/week throughout culture• During expected challenge periods (e.g. summer) increase application rate
• Trial in 7 farms in 2016
Results from Field trials on Polish CarpResuts
19
• 4 farms had no recurring symptoms of previously described diseases
• 1 farm had recurring tapeworm problems which were treated with deworming agent.
• After treatment with algo-bio products tapeworm symptoms were not observed in the spring of 2016
• 3 remaining farms did not follow recommendations
Problems with trout farming in Poland
20
• Reduced feed intake
• Due to poor water quality
• Due Hexamita salmonis• Decreased feeding• Congestion• High mortality
Field trials on Polish trout farms
21
• Difficult to treat because nothing works 100%• Normally deworming treatment is applied• Requires 5-8 applications
• Trout stop feeding after 2nd treatment
• Application Digestsea 1ml/10kg biomass 1x/week
• Trout started feeding within 2 days after application
• Confirmed treatment in 3 farms
Eel farming in Poland
22
• Strengthen eel health • Improve feed efficiency
• Application• 1ml Searup/10kg fish biomass• 1ml Digestsea/10kg fish biomass
• Apply daily during 1st 6 days• Every 2 days after that
• Improved resistance to bacterial and parasite infections
• Improved feed intake• Improved feeding efficiency
• FCR reduced from 1,0 to 0,8
Searup – Tilapia anti transport stress trial
Searup – Tilapia anti transport stress trial
Control1,000 fishNo Searup
0.2% Searup1,000 fish
5 days
No Searup
300fish
No Searup
300fish
TRANSPORT STRESS
PRE-NURSERY STRESS TEST
POST-NURSERY STRESS TEST
PACKING IN BAGS 800 FISH FOR 16 HOURS
Improved survival rates 7 days after stress and searupapplication
Searup – Tilapia anti transport stress trial
95
78
97
84
50556065707580859095
100
Immediately after After 7 days
Survival rate after stress (%)
Control Searup 5d
+7.7%
Larval antistress and EMS challenge trial – L. vannamei
All PLs tested negative to WSSV, YHV, TSV, MBV and V. parahaemolyticuscausing EMS by PCR method Shrimp PL5 raised in 4 (500L) tanks at 50pcs/L
Treatment 1: feed with standard diet.Treatment 2: feed with Searup (0,1%) during 3 consecutive days prior to
stress tests.Treatment 3: feed with Searup (0,1%) during 5 consecutive days prior to
stress tests.Treatment 4: feed with Searup (0,1%) during 7 consecutive days prior to
stress tests.
At stage PL12 shrimp were transferred to 30L tanks where they were submitted to three different stress tests (10ppt and 30ppt salinity, and 100ppm formalin) 4 replicates per test
Searup stress trial – L. vannameiCan Tho University, College of Aquaculture and Fisheries, Dept. Of Aquatic Pathology
Searup stress trial – L. vannamei
10 ppt salinity stress test
0
2
4
6
8
10
12
3h 6h 9h 24h
Cum
mul
ativ
e m
orta
lity
(%)
Hours post exposure to 10ppt salinity
Cummulative mortality post stress challengecontrol 3d Searup
0
2
4
6
8
3h 6h 9h 24h
Cum
mul
ativ
e m
orta
lity
(%)
Hours post exposure to 30ppt salinity
Cummulative mortality post stress challenge
control 3d Searup
-1
1
3
5
7
9
3h 6h 9h 24h
Cum
mul
ativ
e m
orta
lity
(%)
Hours post exposure to 100ppt formalin
Cummulative mortality post stress challengecontrol 3d Searup
30 ppt salinity stress test
100ppm formalin stress test
Searup EMS challenge trial – L. vannamei
All PLs tested negative to WSSV, YHV, TSV, MBV and V. parahaemolyticus causing EMS by PCR method Shrimp PL5 raised in 4 (500L) tanks at 50pcs/L
Treatment 1: feed with standard diet.Treatment 2: feed with Searup (0,1%) during 3 consecutive days, 22d before challenge.Treatment 3: feed with Searup (0,1%) during 5 consecutive days, 20d before challenge.Treatment 4: feed with Searup (0,1%) during 7 consecutive days, 18d before challenge.
At stage PL25 shrimp were transferred to 30L tanks (500pcs/L) where they were submitted to an immersion challenge with EMS causing bacteria (Vibrio parahaemolyticus).
4 replicates per test
Can Tho University, College of Aquaculture and Fisheries, Dept. Of Aquatic Pathology
Searup EMS challenge trial – L. vannameiEMS/AHPND challenge test
0
10
20
30
40
50
60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cum
mul
ativ
e m
orta
lity
(%)
Days post challenge
Cummulative mortalities after AHPNS challenge
Neg cont
Pos cont
3d Searup
5d Searup
7d Searup
• Extract from naturally harvested seaweeds• High bioactive polysaccharides• Lab trials and field trials showing improved
performance • Successfully tested on multiple species
Seaweed sulphated polysaccharidesConclusions
Searup strengthens natural defenses for improve perform
Pioneering theuses of algae
Thank you
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