http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/ FAO Second International Technical Seminar/Workshop on Acute hepatopancreatic necrosis disease (AHPND) There is a way forward! FAO Technical Cooperation Programme: TCP/INT/3501 and TCP/INT/3502.

1. AHPND/EMS: Interaction,
control and pond management
Pikul Jiravanichpaisal
pikul.j@fishvetgroup.com
1

2. The digestive tract is a first entry point for many pathogens
Gregarines
Shrimpcenter.com Photo credits
White Feces Syndrome
AHPND/Early Mortality Syndrome (EMS)
Vibriosis
or
EHP WSSV
2

3. 3

4. 4

5. The shrimp defense mechanism: simple but efficient

6. Shrimp GI Tract Physical and biochemical
barrier
6

7. LectinsProPO
Antimicrobial peptides
Clotting protein Phagocytosis
Ecapsulation/
nodule formation
Pattern recognition protein
Antiviral
defense

8. Cellular Immunity (Haemocytes)
Bac
He

9. What's the difference between Vibriosis and AHPND ?
V. harveyi -infected HP AHPNDVp -infected HP
9

10. Uninfected shrimp-upper midgut Infected shrimp-upper midgut
Anterior midgut and
anterior midgut cecum
are also targeted for
APHNDVp infection
10

11. The junction of stomach and midgut
11
AHPNDvp-6 hours
Control
Lumen
Lumen
Lumen

13. 13

14. Control
6 hours
12 hours
Stomach Hepatopancreas
Differentially expressed transcripts in stomach
of Penaeus monodon in response to AHPND
infection analyzed by Ion torrent sequencing
Soonthornchai W. et al.2016:
Development and Comparative Immunity (accepted)
14

15. Ion PGM sequencing
 1,514 unique genes of significantly DEGs between control and challenge group
 141 unique genes are involved in immune system.
• antimicrobial peptides
• signal transduction
• proPO system
• Oxidative stress
• proteinases/proteinase inhibitors
• apoptotic tumor-related protein
• pathogen recognition immune regulator
• blood clotting system
• adhesive protein
• heat shock protein
15

16. Significant differentially expressed contigs of the stomach cDNA transcriptome
Functions
The number of
genes
The highest expressed genes
Up-
regulation
Down-
regulation
Up-regulation Down-regulation
Antimicrobial peptide 3 2
Anti-lipopolysaccharide factor isoform 6
(P. mododon)
Crustin-like antimicrobial peptide
(P. monodon)
proPO system 16 3
Clip domain serine proteinase 3
(P. triberculatus)
Serine proteinase stubble
(Camponotus floridanus)
Oxidative stress 7 3
Kelch-like ECH-associated protein 1
(Z. nevadensis)
thioredoxin-like protein-like protein
(Scylla paramamosain)
Proteinases/proteinase
inhibitors
8 4
Caspase 4
(L.vannamei)
Alpha 2 macroglobulin
(F. chinensis)
Apoptosic tumor-related protein 7 4
Translationally controlled tumor protein
(P. monodon)
Apoptosis inhibitor
(M. rosenbergii)
Pathogen recognition immune
regulator
9 1
C-type lectin 4
(F. chinensis)
C-type lectin 4 precursor
(Bombyx mori)
16

17. Significant differentially expressed contigs of the stomach cDNA transcriptome
(cont.)
Functions
The number of
genes
The highest expressed genes
Up-
regulation
Down-
regulation
Up-regulation Down-regulation
Blood clotting system 2 3
Clottable protein
(P. monodon)
Hemolymph clottable protein
(L. vannamei)
Adhesive protein 4 0
Nischarin
(Z. nevadensis)
-
Heat shock protein 8 0
Heat shock protein 21
(M. rosenbergii)
-
Signal transduction 21 9
Mitogen-activated protein kinase kinase
kinase 7-interacting protein 1
(Z. nevadensis)
Toll-7
(Nilaparvata lugens)
17

18. 1. Quorum sensing (QS)
• Biofilm
• Virulence factors
2. Adhesive factor
3. Generation time is short
4. Produce Chitin-binding
protein and Chitinase
~2 hours
~9-11 min
V. parahaemolyticus is a versatile pathogen
18

19. 19

20. Full sequence of the AHPND-associated plasmid pVA1.
Chung-Te Lee et al. PNAS 2015;112:10798-10803
©2015 by National Academy of Sciences
20

21. 0.00
20.00
40.00
60.00
80.00
100.00
120.00
8.53x105 2.97x105 5.47x105 6.06x105 9.53x106 1.14x106 6.67x105
Control Vp strain1 Vp strain2 Vp strain3 Vp strain4 Vp strain5 Vp strain6 Vp strain7
%Mortality
AHPND-causing Vp
Immersion Challenge with different Vp strains
24 h 48 h 72 h 96 h
Q: Is only PirA and PirB a deadly toxin?
21

22. Control Non-AHPND Vp AHPND-causing Vp
30 min
60 min
22

23. RESERVOIR
Water/soil
Sick shrimp
Feed residues
PORTAL of EXIT
GI tract
METHOD of
TRANSMISSION
Food
water
PORTAL of ENTRY
GI tract
HOST
A susceptible
shrimp
CAUSATIVE AGENT
Vp
CHAIN of INFECTION
Chain of VP infection
23

24. Sick shrimp
crustacean
Biofilm
Planktonic V. para
Residue feed
The reservoir of Vp
in shrimp pond
24

25. Ways to break the chain of Vp infection without using antibiotics
25

26. 1. Non-sustainable approach: Antibiotics
2. A sustainable strategy: Probiotics, Phage therapy
QS disruption
Strategies for disease prevention and control
26

27. (Live) Feed
Water treatment
- Biofilters
- Bioremediation
- Oxidation (ozone, UV)
Environment
- Temperature, dissolved oxygen,
salinity, …..
- Organic matter & nutrients
Host: Gut microbiota:
- Immunostimalation - Probiotics/prebiotics (antagonism)
- Natural antimicrobial compounds
- Quorum sensing disruption
- Microbial maturation
- Probiotics (antagonism)
- Natural antimicrobial compounds
- Quorum sensing disruption
- Phage therapy
- Disinfection (ozone, UV)
Water microbiota
- Beneficial
- Specific/opportunistic
pathogens
- Neutral
Host health, growth and disease resistance
Gut microbiota Host
The interaction between the environment, shrimp, and the microorganisms in aquaculture system
Modified from its original
version (De Schryver et al.
2012. Infectious disease in
Aquaculture. pp 394-418)
27

28. Contribution to
digestion
Water quality
improvement
Antagonistic
compound
production
Competition
for adhesion sites
Competition
for nutrients
Immune
stimulation
Host
Pathogen
ProbioticsProbiotics
Overview of the modes of action of probiotics
Modified from its original version (De Schryver et al. 2012. Infectious disease in Aquaculture. pp 394-418)
28

29. 4-5 hours
Gastrointestinal transit time Human VS Shrimp
Gastrointestinal transit time is the interval time between ingestion of food and it’s elimination as feces.
10 h to
several days
3-5 h
2-4 h
29

30. The intestine and its microflora are partners for the protection of the host
Intestine Mucosal barriers Local immune system Microflora
www.visualphoto.com www.ecdu.europa.eu Photo credits
30

31. What is the role of the gut microbiota in shrimp health?
31

32. 32

33. • Narrow-spectrum antimicrobial/ high specificity
• Affect only their target pathogen and not microflora
• For prevention
• safe
Bacteriophage as biological control agents in shrimp culture
33

34. https://sites.tufts.edu/quorumsensing/quorumsensing101/
What types of behaviors are regulated by QS?
Quorum sensing disruption
34

35. 70%
25%
5%
Khun Somchai Lerkpookee
Settlement and
sedimentation area
Water treatment, recycling
and storage area
Culture area
Changing farm structure:
Better Management Practices: Clear water – shrimp grow-out culture system
35

36. Pond sides and bottom are lined with PE
Organic matter and sediment must be treated and removed
36

37. Water is recycled as much as possible to reduce biosecurity risk
37

38. • Filter out carriers
• Precipitate the organic matter
• Reduce microorganism load
38

39. Recycled water should be clear (low in organic matter)
39

40. • Biosecurity (water, feeds, seed, people,
equipment, animals, etc.) remains critical
• A higher stocking density (2 million per hectare
or higher) is possible
On site shrimp nursing is conducted
40

41. G. Bergsten et al. / International Journal of
Medical Microbiology 295 (2005) 487–502
The virulence tends to decrease with time
41

42. Entercytozoon hepatopenaei EHP

43. H&E Giemsa Masson’s trichrome (modified)
How to detect EHP:
• Wet-mount
• Histology technique
• In situ hybridization
• PCR
43

44. Target organs for infection with EHP Larvae-midgut
Juvenile-midgut
• Hepatopancreas
• Midgut
Hepatopancreas
44

45. Post larvae15

46. Co-infection (Bacteria and EHP)
46

47. Infected larvae have been kept at -20◦C for one month
One gram shrimp fed with EHP-infected Larvae
Portal of Entry
47

48. Acknowledgements
Prof. Kenneth Söderhäderhall, Uppsala University
Dr. Sage Chaiyapechara, Biotec
Dr. Wipasiri Soonthornchai, Biotec
Khun Don Griffith, FVG Asia
Dr. Anndy Shinn, FVG Asia
Khun Pacharaporn Angthong, FVG Asia
Khun Somchai Lerkpookee

49. THANK YOU