Effect of an immuno-modulating algal extract on disease prevention in aquaculture

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1 Effect of an immuno-modulating algal extract on disease prevention in aquaculture Maarten Jay van Schoonhoven, Aqua Care Manager Olmix

2 Why Seaweeds: Natural Bioactive Renewable Blue biotechnology from the sea

3 Common seaweeds FSC guide

4 Algae Seaweed Classification Phaeophyta (Brown Seaweeds) 1700 species Prodominantly brown due to carotenoid fucoxanthin Primary polysaccharides: alginates, laminarins, fucoidans and cellulose 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 4

5 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, Vitamins Pigments - Magnesium, Sulfur, Phosphorous, Chlorine, Manganese - Many other trace elements. - A, B1, B2, B6, B12, C, D3, E, K Phycoerythrin (antioxidant) Carotenoids

6 SULPHATED POLYSACCHARIDES MSP Chemically and biologically active Marine Sulfated Polysaccharides substances used in biochemistry and medicine. Unique chemical compositions With different structural compositions depending on their origin (brown, green, red ) The new focus on sulfated polysaccharides and their biopharmacological activities is due to their immuno-modulatory, antihyperlipidemic, antiviral, antibacterial and anti-tumor effects.

7 Unbranched homo-polysaccharide Branched homo-polysaccharide Sugar unit Branched sulfated hetero-polysaccharide Sulfate S S S Branched heteropolysaccharide S S S

8 Linear polysaccharide Sugar unit Branched sulfated hetero-polysaccharide 1 - Diversity of sugar units (e.g: glucose, fructose) 2 - Presence of rare sugars (e.g: rhamnose, fucose) 3 - Branching Increased biological activities 4 - 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 Homo-polysaccharide ramifié Sulfate S S S Branched heteropolysaccharide S S S

9 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

10 Algal active ingredients properties: Biological activities Marine 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

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12 TEST 1. Intestinal Porcine Epithelial Cells (IPEC-1) s differentiation and stimulation of the production of «Messenger molecules» of the immune system Cell culture : 10 to 14 days with Dexamethasone (10-7 M) 0,25x10 5 IPEC-1 cells/cm 2 3 days of cell culture, until the observation of confluence Trans epithelial resistance, immunohistochemistry, and microscopic observations Addition of the MSP at different concentrations Analyze of mediator gene expression by RT-qPCR

13 Expression relative RESULTS CYTOKINES AND CHEMOKINES S EXPRESSION IN IPEC-1 EPITHELIAL CELLS CCL20 Lymphocytes and dendritic cell s recruitment ** 8, IL-8 Neutrophils' recruitment & phagocytosis ** 11, 4 TNFα Phagocytosis & neutrophils chemiotaxis ** 6, IL-1α B Lymphocytes: proliferation and differentiation. ** 6,2 0 DMEM LPS Dose (mg/ml) 0 DMEM LPS Dose (mg/ml) DMEM LPS Dose (mg/ml) 0 DMEM LPS Dose (mg/ml) 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) 0

14 MSP Immunomodulating MSP 1. MSP binding on the receptors Destruction of the microbe before a strong proliferation 2. Receptor activation Receptors (TLR, MR, LR ) 4. Leucocyte activation NK, macrophages = Innate Immunity Lymphocytes T, B = Adaptive immunity Epithelial cell Transcription Nucleus Signal Transduction 3. Cell function activation Production of : - Cytokines (e.g: TNFα, IL-1, IL-6 ) - Chemokines (e.g: CCL2, CCL20 ) Hayashi et al., 2003 ; Makarenkova, I.,2012

15 Use of Searup and Digestsea in freshwater fish production in Poland 15

16 Common diseases in Polish Carp production 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 16

17 Common diseases in Polish Carp production 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 Bothriocephalus ageilognathi) most common in summer Few symptoms, other than reduced feed intake and poor growth 17

18 Field trials on Polish carp 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

19 Results from Field trials on Polish Carp Resuts 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 remaining farms did not follow recommendations 19

20 Problems with trout farming in Poland Reduced feed intake Due to poor water quality Due Hexamita salmonis Decreased feeding Congestion High mortality 20

21 Field trials on Polish trout farms 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 21

22 Eel farming in Poland Strengthen eel health Improve feed efficiency Application 1ml Searup/10kg fish biomass 1ml Digestsea/10kg fish biomass Apply daily during 1 st 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 22

23 Searup Tilapia anti transport stress trial

24 Searup Tilapia anti transport stress trial PRE-NURSERY STRESS TEST Control 1,000 fish No Searup 0.2% Searup 1,000 fish 5 days TRANSPORT STRESS PACKING IN BAGS 800 FISH FOR 16 HOURS POST-NURSERY STRESS TEST No Searup 300 fish No Searup 300 fish

25 Searup Tilapia anti transport stress trial Survival rate after stress (%) Immediately after +7.7% After 7 days Control Searup 5d Improved survival rates 7 days after stress and searup application

26 Larval antistress and EMS challenge trial L. vannamei

27 Searup stress trial L. vannamei Can Tho University, College of Aquaculture and Fisheries, Dept. Of Aquatic Pathology 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 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

28 Searup stress trial L. vannamei Cummulative mortality (%) ppt salinity stress test Cummulative mortality post stress challenge control 3d Searup Cummulative mortality (%) ppt salinity stress test Cummulative mortality post stress challenge control 3d Searup Cummulative mortality (%) ppm formalin stress test Cummulative mortality post stress control challenge3d Searup 0 3h 6h 9h 24h Hours post exposure to 10ppt salinity 0 3h 6h 9h 24h Hours post exposure to 30ppt salinity -1 3h 6h 9h 24h Hours post exposure to 100ppt formalin

29 Searup EMS challenge trial L. vannamei Can Tho University, College of Aquaculture and Fisheries, Dept. Of Aquatic Pathology 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

30 Searup EMS challenge trial L. vannamei EMS/AHPND challenge test Cummulative mortalities after AHPNS challenge Cummulative mortality (%) Neg cont Pos cont 3d Searup 5d Searup 7d Searup Days post challenge

31 Seaweed sulphated polysaccharides Conclusions Extract from naturally harvested seaweeds High bioactive polysaccharides Lab trials and field trials showing improved performance Successfully tested on multiple species Searup strengthens natural defenses for improve perform

32 Pioneering the uses of algae

33 Thank you