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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