recent developments in global shrimp health management...shrimp health/microbial management...
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Recent Developments in Global Shrimp Health
Management
AQUA2018, August 25-29 2018; Montpellier, France
FORUM: PRODUCTION OF PENAID SHRIMP IN EUROPE - RISKS AND OPPORTUNITIES
Kartik BARUAH Department of Animal Production & Management
Swedish University of Agricultural Sciences, Uppsala, Sweden
Peter BOSSIER, Parisa NOROUZITALLAB, Tom DEFOIRDT, Patrick SORGELOOS Lab of Aquaculture & Artemia Reference Center, Ghent University, Belgium
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Shrimp Aquaculture Production
The expansion of farmed shrimps is not without diseases
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Early Mortality Syndrome in Shrimps – The Perfect Killer
IMPACT: > billion dollar loss
Countries affected: South America, China, Vietnam, Thailand
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Control Measures for Bacterial Diseases in Aquaculture Animals
DISEASE CONTROL STRATEGIESGood husbandary
Probiotics
Immunostimulants
Vaccines
Acidifiers
NOT BROADSPECTRUM LACK OF CONSISTENCY
KEY FOR EFFICACY
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Controlling Diseases in Human – Practice Followed
ISOLATION & INTENSIVE CARE
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Controlling Diseases in Terrestrial Animals – Practice Followed
ISOLATION & INTENSIVE CARE
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Role of Environment in Host-Pathogen Interaction
HOST(Human, cow)
SPECIES INTERACTION
PATHOGEN
ENVIRONMENT
REGULATON OF DISEASE OUTCOME
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The 3-Dimensional Aquaculture Environment
External Internal
Defense system
Gut flora
Environmental factors
(pond water)
Microorganisms
Diet
Stress
Multiple agents interact to cause diseases in aqaculture animals
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Multiple Agents Cause Diseases in Aquaculture Animals
External Internal
Defense system
Gut flora
Environmental factors
(pond water)
Microorganisms
Diet
Stress
• Inconsistency of anti-infective strategy – Single disease target
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A Paradigm Shift in Shrimp Health Research
Aquaculture Stress/Health Management
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Designing of Anti-infective Strategy
HOST
PATHOGENSHolistic Approach
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Critical Factor in Host-Pathogen Interaction Studies
Model organism
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Virulence factor activity
Inhibition of growth
Under laboratory conditions: no risk for production, more control, yet labor intensive
Under real production conditions: trial and error,
uncontrolled, risk for production
13 22/10/2015
Intermediate step:
GART, the Gnotobiotic Artemia
screening platform
Test System - Development of Disease Control Strategies
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Shrimp Health and Microbial Management
Heat Shock Protein
Probiotics
Glucans
Polyphenols
Organic acids
BioflocEpigenetics
Quorum Sensing
Shrimp Health/Microbial Management
Genetics
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Findings Snapshot
Heat Shock Protein
Probiotics
Glucans
Polyphenols
Butyric acid
BioflocEpigenetics
Quorum Sensing
Shrimp Health/Microbial Management
Genetics
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Quorum Sensing
Shrimp Health & Microbial Mgt: Targeting the Pathogen
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AHA!
• Before: bacteria = Separate entities
Quorum Sensing
• Now: bacteria sense and respond to environmentand to each other
o Extracellular signal molecules
o ≈ hormones in higher organisms
• QS in vibrios: multi-channel systems:
• Documented in:• V. alginolyticus
• V. anguillarum
• V. campbellii / V. harveyi
• V. ichthyoenteri
• V. mimicus
• V. parahaemolyticus
• V. salmonicida
• V. scophthalmi
• V. vulnificus
QUORUM SENSING SYSTEMS
LuxSLuxQ
LuxP
LuxM
LuxN CqsS
CqsA
LuxU
LuxO
LuxR
sRNA’s + Hfq
σ54
NO
OH
H OH O
HAI-1AI-2
OH
O
CAI-1
Promoter of
target genes
OOB
-
O
OH
OH
OH OH
• Virulence factors• In vitro activity assays
• In vitro gene expression (RT realtime PCR)
QS-REGULATED PHENOTYPES
Quorum sensing
Metalloprotease
“Extracellular toxin”
Type III secretion system
Siderophore
Phospholipase
Chitinase
+
-
Chitinase assay
More info: Ruwandeepika et al. (2012) Reviews in Aquaculture 4: 59-74
• Brine shrimp (Artemia): model organism for shrimp
• Creating gnotobiotic cultures starting with axenic
larvae (Instar II nauplii)
Only bacteria added to cultures are present!
Brine shrimp larva
INTERACTION: Vibrio – brine shrimp
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Artemia Model in Bacterial QS Research
BB120 PN9801
Art
em
ia s
urv
iva
l (%
)
0
20
40
60
80
100
Fur 0 mg/l
Fur 5 mg/l
Fur 10 mg/l
Fur 20 mg/l
Fur 50 mg/l
Furanone
is toxic at
50 mg/l
BB120 LMG21363
20 mg/l
furanone:
protection
against both
vibrios
• In vivo challenge tests with gnotobiotic Artemia and weak (BB120) or more
virulent (LMG21363) strain
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Disruption of QS to Control Bacterial Infections
• Use of QS inhibitors (e.g. plant extracts/plant-derieved compound)
[Furanone] (mg/l)
0,01 0,1 1 10 100 1000
Bio
lum
inescence (
%)
0
20
40
60
80
100
120
140
JAF375 (CAI-1)
JMH597 (AI-2)
JMH612 (HAI-1)
0.01 0.1 1 10 100 1000
• Effect of the furanone on bioluminescence of V. harveyi
The furanone blocks all three channels
of the quorum sensing system
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Time (h)
0 5 10 15 20 25 30
OD
600
0,01
0,1
1
10
BB120
BB120 + Fur 50 mg/l
• In vitro growth of luminescent vibrios without and with furanone (50 mg/l)
The furanone does not affect growth
of the vibrios
Disruption of QS to Control Bacterial Infections
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Epigenetics
Shrimp Health & Microbial Mgt: Targeting the Host
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Teaching Shrimps Self-Defence to Fight Infections
Shrimp Health & Microbial Mgt: Targeting the Host
(clonal population)
Parthenogenetic Artemia
TF1 CF1
TF2 CF2
TF3 CF3
ISO
THERMIC
Experimental Design
Common garden test - Verifying stress-resistant phenotypes
42°C for 15 min
Or
Vibrio campbellii 107 cells/ml
20 instar II nauplii in 6 replicates
F1 F2 F3
Thermo-tolerance test
******
*** *** *** ****** ***
0
0,2
0,4
0,6
0,8
1
0 3 6 9 12 15 18 21 24 27
Pro
po
rtio
n o
f th
e s
urv
ive
d a
nim
als
Time (h)
TF1
CF1
*** ***
*** ***
*** *** ******
** **
0
0,2
0,4
0,6
0,8
1
0 3 6 9 12 15 18 21 24 27
Pro
po
rtio
n o
f th
e s
urv
ive
d a
nim
als
Time (h)
TF2
CF2
*
* *
* * * *
0
0,2
0,4
0,6
0,8
1
0 3 6 9 12 15 18 21 24 27
Pro
po
rtio
n o
f th
e s
urv
ive
d a
nim
als
Time (h)
TF3
CF3
TF1 > CF1 TF2 > CF2
TF3 > CF3
***
***
0
0,2
0,4
0,6
0,8
1
0 12 24 36 48
Pro
po
rtio
n o
f th
e s
urv
ive
d a
nim
als
Time (h)
TF1 CF1
***
***
0
0,2
0,4
0,6
0,8
1
0 12 24 36 48
Pro
po
rtio
n o
f th
e s
urv
ive
d a
nim
als
Time (h)
TF2 CF2
**
***
0
0,2
0,4
0,6
0,8
1
0 12 24 36 48
Pro
po
rtio
n o
f th
e s
urv
ive
d a
nim
als
Time (h)
TF3 CF3
TF1 > CF1TF2 > CF2
TF3 > CF3
V. campbellii resistance
HSP-INDUCING COMPOUNDS
IN HEALTH & DISEASE
Shrimp Health & Microbial Mgt: Targeting the host-pathogen
Function of Intracellular Hsp
Unfolded
Misfolded
Correctly folded protein
Hsp
Hsp
Conserved proteins acting as molecular chaperones - bind to unfoldedproteins (nascent polypeptides or denatured ones) - facilitate theirrefolding to the native state
Involved in protein translocation and degradation
Immunogenic proteins that modulate both innate and adaptive immuneresponses
Hsp-inducing Compound in Shrimp Health Management
Pyrogallol
Carvacrol
Phloroglucinol
Protective Effect of Phloroglucinol against V. parahaemolyticus
i
a
b
d
f
h
g
e
c
0
20
40
60
80
100
Negativecontrol
Positivecontrol
5 µM 10 µM 20 µM 30 µM 40 µM 50 µM 100 µM
Surv
ival
(%
)
Treatments
Negative control: No pretreatment, no challenge
Positive control: No pretreatment, Vibrio challenge
“Whole-animal Response”
Kumar et al. 2018. Frontiers in Immunology. 9:1091
Mode of Action of Phloroglucinol: Induction of Hsp70
c
a
b
0
0,2
0,4
0,6
0,8
1
HeLa cells Control Phloroglucinol
Re
lati
ve p
rod
uct
ion
of
Hsp
70
in b
rin
e
shri
mp
larv
ae
Treatments
M HeLa cells Control Phloroglucinol
75
Kumar et al. 2018. Frontiers in Immunology. 9:1091
In vivo knockdown of Artemia Hsp70 using RNAi
Figure: Knockdown of hsp70 mRNA in brine shrimp larvae. Equal amounts of RNA from
brine shrimp larvae were amplified by RT-PCR and the products were resolved by
electrophoresis in agarose gels.
M - 100bp DNA ladder,
Lane 1 - Wild type brine shrimp larvae,
Lane 2 - Brine shrimp larvae injected with dsRNA gfp
Lane 3 - Brine shrimp larvae injected with dsRNA hsp70
Lane 4 - Wild type brine shrimp larvae pretreated with phloroglucinol
Lane 5 - Brine shrimp larvae injected with dsRNA gfp and pretreated with phloroglucinol,
Lane 6 - Brine shrimp larvae injected with dsRNA hsp70 and pretreated with phloroglucinol
*
0
20
40
60
80
100
Hsp70- (dsRNAHsp70)
Hsp70+ (dsRNA Gfp) Hsp70- (dsRNAHsp70)
Hsp70+ (dsRNA Gfp)
Positive control Phloroglucinol
Surv
ival
(%
)
Treatments
M WT dsRNA gfp Hsp70(-) WT+P WT+gfp Hsp70(-) + P
Phloroglucinol induces anti-Vibrio effect by inducing Hsp70
Kumar et al. 2018. Frontiers in Immunology. 9:1091
Mode of Action of Phloroglucinol
Damage Control System
(Hsp70)
+
-
Reduce virulence factor
production in Vibrio
Kumar et al. 2018. Frontiers in Immunology. 9:1091
Phloroglucinol
Anti-Vibrio Effect of Phloroglucinol – Validation Study Ongoing
The Health Beneficial Activities of Tex-OE®
Findings Snapshot
Temperature stress
Osmotic shock
V. campbellii
V. harveyi
Baruah et al. (2014) Dev & Comp Immunol 46:470-9
Baruah et al. (2012) Aquaculture 334-337:152-158
FUTURE PERSPECTIVES
MODE OF ACTION
Polyphenols
Heat Shock Protein
Quorum Sensing Disruption
OMNIBIOTIC -A HOLISTIC STRATEGY
Probiotics
BROADSPECTRUM CONSISTENT
ACKNOWLEDGMENTS
thanK yoU
BOF-Special Research Fund