selection study of potential probiotic bacteria for shrimp ... 5 - thursday/12.00-12.20... · larvi...

larvi 2013

ghent university, belgium, 2-5 september 2013

6th fish & shellfish larviculture symposium

Selection study of potentialprobiotic bacteria for shrimp hatcheries

in New Caledonia

Dominique Pham

Selection study of potentialprobiotic bacteria for shrimphatcheries in New Caledonia

Pham Dominique, Ansquer Dominique, Chevalier Anne, Peyramale Aude,Dauga Clément, Wabete Nelly and Labreuche Yannick.

Unité de Recherche Lagon, Eco-système et Aquaculture Durable,

Ifremer

BP 2059, Nouméa - New Caledonia

Larvi 2013, September 2nd-5th, 2013, Ghent, Belgium

Shrimp farming in New Caledonia

• Commercial production since 1983

Litopenaeus stylirostris

• Semi-intensive culture from Litopenaeus stylirostris captivebroodstock

• 2500 tons in 2005, less than 1200 tons in 2010

• 2 seasonal Vibrio pathogens in grow-out ponds(V. penaeicidae and V. nigripulchritudo)

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nd

tem

per

atu

re (

°C)

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mortality

Temperature

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Po

nd

tem

per

atu

re (

°C)

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mortality

Temperature

Cold seasonCold season

Hatchery constraints in New Caledonia

• Most of postlarvae demand concentrated in warm season(october to january)

• Increasing use of antibiotics to stabilize production

• Research project for evaluating alternativeprophylactic treatments in hatchery such asprobiotics

January April-May September-October

December

Weak survival in grow-out ponds

Warm T°C Warm T°CCold T°C

Experimental approach

Potential probionts selection

Strains characterization / identification

In vitro growth-inhibition test

Innocuousness test

Effectiveness test in larval rearing

Impact on animal status

Potential probionts selection

Screening for antagonistic activity againstV. nigripulchritudo.

Selection of 7 isolates :NC72, NC197, NC201, NC203, NC204,

NC257 and NC297IPNC/Ifremer

IPNC/Ifremer

493 isolates sampled in extreme marine environments in New Caledonia

Strains characterization / identification

5 isolates : Pseudoalteromonas genus

NC201 close to P. maricaloris

NC257,NC297, NC203, NC204,

NC197 close to P. piscicida

Several species with antibacterial, antifungaland antialgal properties

1 isolate : Vibrio genus

NC72 belonging to Harveyi clade

Bacteria group including pathogens andprobiotic strains for marine invertebrates

V.penaeicida (T) DSM 14398T AJ421444

V. fortis (T) type strain: LMG 21557 AJ514916

V. mytili (T) X99761

V. diabolicus (T) HE800 X99762

V. ponticus (T) type strain: CECT 5869 AJ630103

72

V. parahaemolyticus (T) ATCC 17802 Vp23 AF388386

V. azureus (T) LC2-005 ( NBRC 104587) AB428897

V. rotiferianus (T) type strain:LMG 21460 AJ316187

V. natriegens (T) ATCC 14048T X74714

V. campbellii (T) ATCC 25920 X56575

V. alginolyticus (T) ATCC 17749 X56576

0.001

Phylogenetic analysis

KG Primer

M203 72 204 220 201 257 297 197

AP-PCR

API 20E Gallery Test

Inhibitory test on V. harveyi ORM4-GFP growth

1 2

V. harveyi GFP V. harveyi GFP + Isolate

x 3Fluorescence reading after 48h

0

0,2

0,4

0,6

0,8

1

1,2

1,4

ORM4-GFP ORM4-GFPNC72

ORM4-GFPNC197

ORM4-GFPNC201

ORM4-GFPNC203

ORM4-GFPNC204

ORM4-GFPNC257

ORM4-GFPNC297

Fluo

resc

ence

(arb

itrar

yun

it)

d

cd

cc c

ab

c

a

105 CFU.mL−1

29°C

1 2


x 3Fluorescence reading after48h

- Weak inhibition activity

from NC257

0

0,2

0,4

0,6

0,8

1

1,2

1,4


ORM4-GFPNC197

ORM4-GFPNC201

ORM4-GFPNC203

ORM4-GFPNC204

ORM4-GFPNC257

ORM4-GFPNC297

Fluo

resc

ence

(arb

itrar

yun

it)

d

cd

cc c

ab

c

a

105 CFU.mL−1

29°C


1 2


0

0,2

0,4

0,6

0,8

1

1,2

1,4


ORM4-GFPNC197

ORM4-GFPNC201

ORM4-GFPNC203

ORM4-GFPNC204

ORM4-GFPNC257

ORM4-GFPNC297

Fluo

resc

ence

(arb

itrar

yun

it)

d

cd

cc c

ab

c

a

Excellent antagonistic activity from 6 out of the 7 isolatestowards V. harveyi ORM4-GFP

- Strong inhibition from

NC197,NC201, NC203,

NC204, NC297

x 3Fluorescence reading after48h

- Very strong inhibition

activity from NC72

105 CFU.mL−1

29°C


Harmfulness of probiotic candidates

0%

20%

40%

60%

80%

100%

120%

control NC72 NC197 NC201 NC203 NC204 NC257 NC297

mea

n%

surv

ival

probiotic candidates

Mysis 2

PL9

No survival alteration from any

isolates in both stages

No abnormality in larvae and

postlarvae behaviour with all

isolates

Pathogenicity test towards Mysis 2 and PL9

- Mysis 2 in duplicates for 48 hours

- PL9 in triplicates for 72 hours

- Isolates final concentration of 105 CFU/mL

No pathogenicity detected for any isolates

5,5

6,0

6,5

7,0

0%

20%

40%

60%

80%

100%

NT AB NC72 NC197 NC201 NC203 NC297

Dev

elop

men

t In

dex

Surv

ival

(%

)

Probiotic effectiveness in larval rearing :from nauplius to mysis

- No treatment (NT)- Antibiotic treatment (AB) at D3, D5, D7 and D9 at 2.5g/m3

- 5 probiotic treatments: Vibrio NC72 and Pseudoalteromonas NC197, NC201,NC203, NC297 every day administration at 105 CFU/ml

Zoea 1-3

D6

Mysis1-3

D9D2D0

Nauplius 1-5 PL1-4

x3100 L Survival and growth

180 larvae/L29°C

5,5

6,0

6,5

7,0

0%

20%

40%

60%

80%

100%


Dev

elop

men

t In

dex

Surv

ival

(%

)

a


b ab ab ab ab ab

Similar or better survivalrates with probiotic

treatments


- 5 probiotic treatments: Vibrio NC72 and Pseudoalteromonas NC197, NC201,NC203, NC297 every day administration at 105 UFC/ml

Zoea 1-3

D6

Mysis1-3

D9D2D0

Nauplius 1-5 PL1-4


180 larvae/L29°C

5,5

6,0

6,5

7,0

0%

20%

40%

60%

80%

100%


Dev

elop

men

t In

dex

Surv

ival

(%

)


Better growth withprobiotics comparedto antibiotic

****

** ******

*


- 5 probiotic treatments: Vibrio NC72 and Pseudoalteromonas NC197, NC201,NC203, NC297 every day administration at 105 UFC/ml

Zoea 1-3

D6

Mysis1-3

D9D2D0

Nauplius 1-5 PL1-4


180 larvae/L29°C

5,5

6,0

6,5

7,0

0%

20%

40%

60%

80%

100%


Dev

elop

men

t In

dex

Surv

ival

(%

)


Selection of NC201for further trials


- 5 probiotic treatments: Vibrio NC72 and Pseudoalteromonas NC197, NC201, NC203, NC297 every dayadministration at 105 UFC/ml

D19

Zoea 1-3

D6

Mysis1-3

D9D2D0

Nauplius 1-5 PL1-4


180 larvae/L29°C

NC201 effectiveness in larval rearing :from nauplius to P9

Posology : every day (NC201) vs each other day (NC2011/2)

0,20

0,25

0,30

0,35

0,40

0,45

0,50

0%

20%

40%

60%

80%

100%

NT AB NC201 NC201 1/2

Mea

n dr

y w

eigh

t (

mg)

Surv

ival

bb

a

b

D19

Zoea 1-3

D6

Mysis1-3

D9D2D0

Nauplius 1-5 PL1-4

x3100 L- Survival- Growth

180 larvae/L29°C


0,20

0,25

0,30

0,35

0,40

0,45

0,50

0%

20%

40%

60%

80%

100%

NT AB NC201 NC201 1/2

Mea

n dr

y w

eigh

t (

mg)

Surv

ival

bb

Same survival enhancement of NC201 each other day compared to every day supply

a

b

D19

Zoea 1-3

D6

Mysis1-3

D9D2D0

Nauplius 1-5 PL1-4

x3100 L

180 larvae/L29°C


- Survival- Growth


0,20

0,25

0,30

0,35

0,40

0,45

0,50

0%

20%

40%

60%

80%

100%

NT AB NC201 NC201 1/2

Mea

n dr

y w

eigh

t (

mg)

Surv

ival

MDW (mg)

bb

a

b

NC201 each other day as effective as NC201 every day


0,25

0,30

0,35

0,40

0,45

0%

20%

40%

60%

80%

100%

NT AB NC201 1/2 NC203 1/2 NC201+203 1/2

Mea

n dr

y w

eigh

t (

mg)

Surv

ival

a

b

ab

Single administration vs combination of two probiotics

D19

Zoea 1-3

D6

Mysis1-3

D9D2D0

Nauplius 1-5 PL1-4

x3100 L

180 larvae/L29°C


- Survival- Growth

No survival improvement with probiotics combination


0,25

0,30

0,35

0,40

0,45

0%

20%

40%

60%

80%

100%

NT AB NC201 1/2 NC203 1/2 NC201+203 1/2

Mea

n dr

y w

eigh

t (

mg)

Surv

ival ab

ab

a

b

ab

D19

Zoea 1-3

D6

Mysis1-3

D9D2D0

Nauplius 1-5 PL1-4

x3100 L

180 larvae/L29°C


- Survival- Growth

No improvement with probiotic combination compared to singleadministration


0,25

0,30

0,35

0,40

0,45

0%

20%

40%

60%

80%

100%

NT AB NC201 1/2 NC203 1/2 NC201+203 1/2

Mea

n dr

y w

eigh

t (

mg)

Surv

ival

MDW (mg)


Three parameters were evaluated :Vibrio load in animalAMP gene expressionResistance to salinity stress test

Probiotic impact on animal status

Three parameters were evaluated :Vibrio loadAMP gene expressionResistance to salinity stress test

Estimation of Vibrio concentration at D9 and D19 in animals on TCBS media.

1,E+00

1,E+01

1,E+02

1,E+03

1,E+04

1,E+05

D9 D19

CF

U/a

nim

al

NT AB NC201


Three parameters were evaluated :Vibrio loadAMP gene expressionResistance to salinity stress test

Could this load drop be dueto higher antimicrobialpeptide action ?

Lower Vibrio concentration in animal at D19 with probiotics

1,E+00

1,E+01

1,E+02

1,E+03

1,E+04

1,E+05

D9 D19

CF

U/a

nim

al

NT AB NC201

a

bb

Estimation of Vibrio concentration at D9 and D19 in animals on TCBS media.



Three parameters were evaluated :Vibrio load in animal at D9 and D19Lyzozyme gene expression at D9 and D19Resistance to salinity stress test

Sampling of pooled 20 animals at D9 and pooled 10 animals at D19 for each triplicate

Gene expression profile analysed by quantitative RT-PCR, using EF1 as housekeeping gene.

0,0

0,5

1,0

1,5

2,0

2,5

D9 D19

Rel

ativ

e ex

pres

sion

Lysozyme

NT AB NC201


Three parameters were evaluated :Vibio load in animal at D9 and D19Lyzozyme gene expression at D9 and D19Resistance to salinity stress test

0,0

0,5

1,0

1,5

2,0

2,5

D9 D19

Rel

ativ

e ex

pres

sion

Lysozyme

NT AB NC201

No detection of significant modulation of Lysozyme expression with probiotics

Sampling of pooled 20 animals at D9 and pooled 10 animals at D19 for each triplicate

Gene expression profile analysed by quantitative RT-PCR, using EF1 as housekeeping gene.

Three parameters were evaluated :Vibrio load in animal at D9 and D19AMP gene expression at D9 and D19Resistance to salinity stress test

Direct transfer from 27 ppt to 17-25 ppt in 5 days-oldpost-larvae from three different batches reared with orwithout probiotics-> survival after 24h

0%

20%

40%

60%

80%

100%

120%

1st : 27 to 20 2nd : 27 to 20 3 rd :27 to 17

Surv

ival

NT

O

OOOOOO

OOOOO

OOO

17 ppt

Salinité 45ppt

20 ppt

25 ppt

27 ppt

27ppt


Three parameters were evaluated :Vibrio load in animal at D9 and D19AMP gene expression at D9 and D19Resistance to salinity stress test

Direct transfer from 27 ppt to 17-25 ppt in 5 days-oldpost-larvae from three different batches reared with orwithout probiotics -> survival after 24h

Resistance enhancement of postlarvae with probiotics

0%

20%

40%

60%

80%

100%

120%

1st : 27 to 20 2nd : 27 to 20 3 rd :27 to 17

Surv

ival

NT NC201

O

OOOOOO

OOOOO

OOO

17 ppt

Salinité 45ppt

20 ppt

25 ppt

27 ppt

27ppt


Conclusions• Possible alternatives to antibiotic treatments in Caledonian hatcheries

• Pseudoalteromonas genus is a good probiont candidate

• NC 201 in single administration each other day improves zootechnicalperformances in larval rearing

• Probiotics can improve global health status of postlarvae

• More studies are necessary to optimize the use of probiotics in larval rearing :

• imprinting time

• administration way

• mode of action

• Better knowledge by experimenting :

• bacterial challenge in larval and postlarval phases

• probiotic effect on juveniles and adults

Thank you for your attention

• This research was financially supported by Ifremer, the Provincial Institutionsand the Government of New Caledonia. Thanks to :

• Hatchery staff in Saint-Vincent (F. Broutoi, S. Collet, S. Girard, J.R.Mailliez and J.M. Peignon) for rearing animals

• Institut Pasteur in New Caledonia for collaboration in bioprospection

• “Plate-forme de Recherche pour les Sciences du Vivant” de NC(PFV-NC) for allowing access to RT-PCR facility.

selection study of potential probiotic bacteria for shrimp ... 5 - thursday/12.00-12.20... · larvi...

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