GART as a platform to investigate disease control strategies against AHPND/EMS in shrimp

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1 GART as a platform to investigate disease control strategies against AHPND/EMS in shrimp Aquaculture Ghent University Niu Yufeng, P. De Schryver, A. Madanperuma Arachchilage Pubudu Kumari, P. Sorgeloos, M. Drouillon, P. Bossier

, 2008) 2 22/10/2015 Aqua UGent Faculty of Bioscience

2 Diseases in shrimp farming Losses due to diseases carefully estimated at around US$15 billion in 15 years (Flegel et al., 2008) 2 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

3 Control strategies Antibiotics: no go! Probiotics Quorum sensing interference Listen up my fellow pathogens! We will start attacking when... we are enough in numbers. Start counting! Phage therapy 3 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

Development of disease control strategies Under real production

of growth Under laboratory conditions: no risk for production, more

step: GART, the gnotobiotic Artemia screening platform 4 22/10/2015 Aqua

4 Development of disease control strategies Under real production conditions: trial and error, uncontrolled, risk for production Inhibition of growth Under laboratory conditions: no risk for production, more control, yet labour intensive Virulence factor activity Intermediate step: GART, the gnotobiotic Artemia screening platform 4 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

5 Gnotobiotic Artemia screening platform: pathogenic challenge 1 Decapsulation and disinfection of cysts to remove all bacteria 3 Stocking of sterile Artemia nauplii in sterile seawater 5 Count surviving nauplii; growth; activity;... after 24-48h 2 Hatching Artemia nauplii in sterile seawater 4 Treatment and/or pathogenic challenge (V. harveyi;v. campbellii;...) 5 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

6 Gnotobiotic Artemia screening platform: model Host Simplification Host Microbial environment COMPLEX Environment Known microbial environment GNOTOBIOTIC 6 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

7 Gnotobiotic Artemia screening platform: advantages Small scale Continuous supply High throughput (48-72 h) Highly controllable Realistic response Efficient in vivo screening platform Economic strategy for product development High reproducibility Reliable results Higher success rate of validation in other species (shrimp) 7 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

8 Use of gnotobiotic Artemia in Early Mortality Syndrome research China (2009) Mexico (2013) Vietnam (2010) Thailand (2012) Philippines (2015) Malaysia (2011) AHPND/EMS on a global scale 8 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

stocking in grow-out pond causes up to 100 % mortality

shrimp EMS infected shrimp Empty stomach Full mid gut Full

pigmented hepatopancreas 9 22/10/2015 Aqua UGent Faculty of

9 Use of gnotobiotic Artemia in Early Mortality Syndrome research Characteristics shrimp post-larvae days after stocking in grow-out pond causes up to 100 % mortality annual losses in Asia: > 1 billion $ US (GAA, 2013) Healthy shrimp EMS infected shrimp Empty stomach Full mid gut Full stomach Atrophied pale hepatopancreas Empty mid gut Large pigmented hepatopancreas 9 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

10 Treatments against AHPND/EMS AHPNS has a bacterial etiology and Koch s Postulates have been satisfied in laboratory challenge studies with the isolate, which has been identified as a member of the Vibrio harveyi clade, most closely related to V. parahaemolyticus V. parahaemolyticus is a normal member of the microbial community in aquaculture systems hence difficult to avoid. Application of traditional sustainable strategies may contribute in preventing the disease: Probiotics/Prebiotics Immunostimulation Quorum sensing disruption /10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

11 Using the gnotobiotic Artemia platform in AHPND/EMS research 1 Finding a representative pathogen: isolates obtained from diseased shrimp with AHPND/EMS symptoms: Isolate Origin (year) Confirmed for Koch s postulates AP2/AP3 positive topa sequence analysis PV1 China (2010) V. parahaemolyticus TW01 Thailand (2013) V. parahaemolyticus KM Thailand (2013) V. parahaemolyticus M0605 Mexico (2013) V. parahaemolyticus M0904 Mexico (2013) V. parahaemolyticus M0903 Mexico (2013) V. parahaemolyticus 11 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

12 Using the gnotobiotic Artemia platform in AHPND/EMS research Average survival (%) Challenge tests using the isolates as pathogens in the model: 100 Experiment 1 A a A Experiment 1 90 a a Experiment 2 a a Experiment 2 80 B b BC b B 70 B 60 C b C 10 0 Control control 10*3 3 10*5 5 10*7 7 Control control *5 5 10* PV1 (CFU/ml) Average survival (%) TW01 (CFU/ml) 12 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

13 Using the gnotobiotic Artemia platform in AHPND/EMS research 2 Challenge tests using the isolates as pathogens in the model: Average survival (%) a Experiment Experiment 1 A ab Experiment 2 90 a Experiment 2 B b 80 A a BC b 70 C 60 B B b b C 10 0 Control control 10*3 3 10*5 5 10* Control control 10*3 3 10*5 5 10* KM (CFU/ml) Average survival (%) M0904 (CFU/ml) 13 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

14 Using the gnotobiotic Artemia platform in AHPND/EMS research 2 Challenge tests using the isolates as pathogens in the model: Average survival (%) Experiment Experiment 1 a A Experiment 2 90 Experiment 2 B bc b BC 80 c C Control control 1010*3 3 10*5 10*7 Control control 10*3 10*5 10* M0605 (CFU/ml) Average survival (%) M0903 (CFU/ml) 14 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

Using the gnotobiotic Artemia platform in AHPND/EMS research 3 Verification of the results in a shrimp model: 15 shrimp/treatment X 15 = Shrimp postlarvae pathogen at 10 6 /10 7

15 Using the gnotobiotic Artemia platform in AHPND/EMS research 3 Verification of the results in a shrimp model: 15 shrimp/treatment X 15 = Shrimp postlarvae pathogen at 10 6 /10 7 CFU/ml 300 ml of natural sea water Determine mortality on a daily basis 15 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

Using the gnotobiotic Artemia platform in AHPND/EMS research 3 Applying the isolates as pathogens in a shrimp model system: Challenge with EMS-pathogen PV1 at 10 6

16 Using the gnotobiotic Artemia platform in AHPND/EMS research 3 Applying the isolates as pathogens in a shrimp model system: Challenge with EMS-pathogen PV1 at 10 6 CFU/mL Challenge with EMS-pathogen TW01 at 10 6 CFU/mL 16 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

Which ones to test on shrimp post-larvae?

17 Example: screening for probiotics to control AHPHND/EMS A library of bacili from culture collections and environmental samples. Which ones to test on shrimp post-larvae? 47 Probiotic candidates 17 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

Example: screening for probiotics to control AHPHND/EMS 47 Probiotic candidates 4 most efficient ones against both PV1 and TW01 in the Artemia

18 Example: screening for probiotics to control AHPHND/EMS 47 Probiotic candidates 4 most efficient ones against both PV1 and TW01 in the Artemia model selected for validation 18 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

19 Example: screening for probiotics to control AHPHND/EMS Validation of the 4 probiotic strains in the shrimp challenge model Shrimp post-larvae PL44: 1) Treatment with the bacilli at 10 7 CFU/mL 2) Challenge with EMSpathogen PV1 at 10 6 CFU/mL 19 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

20 Examples of GART based findings Identification of probiotics against V. harveyi for giant freshwater prawn larviculture (Thai et al., Applied Microbiology and Biotechnology 2014, 98, ) Identification of prebiotics against V. campbellii for giant tiger prawn larviculture (Laranja et al. Veterinary Microbiology 2014, 170, ) Identification of prebiotics to be applied in European sea bass culture, Nile tilapia culture, blue mussel culture,... (De Schryver et al. Applied Microbiology and Biotechnology 2010, 86, ; Hung et al. Aquaculture 2015, 446, ; Situmorang et al. unpublished) Identification of Quorum Sensing disrupting compounds of V. harveyi for giant freshwater prawn larviculture (Pande et al. Veterinary 2013, 406, ) /10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

21 Summary: gnotobiotic Artemia system in aquaculture research Pre- and probiotics... Quorum sensing interference Immunostimulants Gnotobiotic Artemia system Performance: survival, growth, challenge test Bacterial composition: Quality / quantity analysis Host/gene expression analysis & identification marker genes Biochemical analyses Fish, crustaceans and shellfish larvae validation 21 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

22 Conclusion: GART has great potential for the development of anti- EMS/AHPNS strategies Anti-EMS strategies GART Validation in shrimp High throughput + realistic response + highly controlled conditions + less labor/time cost 22 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

Our integrated and multidisciplinary approach Life cycle analysis Morphological development Aquatic veterinary medicine Industry Yields & quality Disease Society Environmental monitoring

23 Our integrated and multidisciplinary approach Life cycle analysis Morphological development Aquatic veterinary medicine Industry Yields & quality Disease Society Environmental monitoring Nutritional research Sustainability Food security Microbial management Genomics 23 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University

We provide multidisciplinary expertise, collaborative research, training, innovations and financing options for R&D in the areas of: Training Your company Models and services

24 Bridging Science and Industry Aquaculture Ghent University (Aqua UGent) is one of world s leading research partners for aquaculture. Collaborative research Innovation We group top scientists of Ghent University that are active in different aspects of sustainable aquaculture and the blue economy. We provide multidisciplinary expertise, collaborative research, training, innovations and financing options for R&D in the areas of: Training Your company Models and services Hatchery management Microbial management Genomics & breeding Aquaculture business management Financing options Multidisciplinary expertise 24 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University Aquaculture@UGent.be

25 Different options of collaboration with Aqua UGent Service to industry Such as GART test=fast, low budget, high throughout, no risk Dedicated Research & Development Dedicated team, more investment, higher return (if succeed) Joint applications for subsidies/project... We are flexible for different approaches. Feed nutrition Feed additives Pro/prebiotics Veterinarian medicine Immunostimulants Aquaculture related 25 22/10/2015 Aqua UGent Faculty of Bioscience Engineering Ghent University

26 Acknowledgements: Robins McIntosh (Charoen Pokphand Group Co., Ltd., Thailand) Dr. Fabiano Thompson (Federal University of Rio de Janeiro, Brasil) Dr. James Brock (Moana Technologies, USA) Dr. Bruno Gomez-Gil (CIAD, Mexico) Contacts: Dr. Niu yufeng ( 牛宇峰 ) Yufeng.niu@UGent.be Thank you for your attention

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