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Disease Problems in Prawn Farming

Prof. Toshiaki ITAMI

Department of Biological Production and Environmental Sciences, University of Miyazaki, Miyazaki, Japan

E-mail: itamit@cc.miyazaki-u.ac.jp

University of Miyazaki

Keio University

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Outline of the Lecture

- History and Prawn Production

- Prawn Culture System

- Diseases

- Detection Method

- Prevention

Start of Prawn Culture

Dr. M. FUDINAGA

Aio, Yamaguchi Pref., Japan

The shrimp seed production technique and the shrimp culture system were developed by M. Fudinaga and his colleagues in the early 1960s in Japan after many ground-breaking studies over the preceding 30 years. Their techniques have been spreading throughout the world and have helped the world shrimp culture industry grow enormously.

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Prawn Production

World Prawn Production

0

200,000

400,000

600,000

800,000

1,000,000

1,200,000

19701973

19761979

19821985

19881991

19941997

2000

0

200,000

400,000

600,000

800,000

1,000,000

1,200,000

19701973

19761979

19821985

19881991

19941997

2000

1993 1997

(From Fishstat Plus, FAO)

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Prawn Production in Each Countries

(From Fishstat Plus, FAO)

0

50,000

100,000

150,000

200,000

250,000

300,000

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

19881989

19901991

19921993

19941995

19961997

19981999

2000

China

Indonesia Viet Nam

217,994

138,000

69,433

0

50,000

100,000

150,000

200,000

250,000

300,000

Thailand299,700

1997

1994

1998

0

50,000

100,000

150,000

200,000

250,000

300,000

1988

1989

1990

1991

1992

1993

1994

1995

19961997

1998

1999

2000

Thailand299,700

0

50,000

100,000

150,000

200,000

250,000

300,000

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

China 217,994

0

500

1,000

1,500

2,000

2,500

3,000

198819

891990

1991

1992199

31994

1995

1996

1997

1998

1999

2000

2,066Japan

Prawn Production in Japan

0.7% of total shrimp production in Thailand

(From Fishstat Plus, FAO)

1994

1994

5

0%

50%

100%

Total Export Quantity = 1,116,284 tons in 2000

EU (34.6%)

USA (25.2%)

Japan (22.1%)

Others (18.1%)

World Trade of Shrimp

(From Fishstat Plus, FAO)

Prawn Culture

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Litopenaeus vannameiP. vannamei(pacific white shrimp)

Penaeus monodonP. monodon(black tiger prawn)

Marsupenaeus japonicusPenaeus japonicus(kuruma prawn)

NEWOLD

Major Target Species of Penaeid Shrimp

Marsupenaeus japonicus

Litopenaeus vannamei

Penaeus monodon

from AAHRI Newsletter

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1 kg of live kuruma shrimp = 5,000 - 12,000 yen(45 – 110 USD)

1 kg of fresh puffer fish = 2,000 - 4,000 yen

(18 – 36 USD)

1 kg of fresh red sea bream = 800 - 1,500 yen (7 – 14 USD)

exchange rate: 110 yen/USD

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9

Sedimentation tank 1 Bio-filter

1.5ｍ

2 ｍ

1.5ｍ

Indoor Culture System

UV Ｐ

Pump（2.2ｋｗ×２）

Ｏ2 generator

10 ｍ

9 ｍ

Operation unit

Breeding tanks

Sedimentation tank 2

Breeding tanks

Research Institute of Land Aqua Culture Technology, Japan

Breeding tanks

Bio-filterSedimentation tank 1 Breeding tanks

Sedimentation tank 2

Indoor culture system for kuruma shrimpResearch Institute of Land Aqua Culture Technology, Japan

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DiseasesViral Diseases

-WSSV-TSV-YHDV-IHHNV

Bacterial Disease- Vibrio harveyi

White Spot Syndrome Virus (WSSV)

- a double stranded DNA virus

- genus whispovirus, family nimaviridea

- rod-shaped , enveloped virus

- 80-120 × 250-380 nm

- genome size: ~ 300,000 bp (292,967-307,287)

- Asia, USA, Central & South America

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Economic losses

China in 1994: 400,000,000 USD

Thailand in 1996: 500,000,000 USD

Ecuador in 1999: 150,000 people lost job

in 2000: 580,000,000 USD

Unknown 35.4 %

1992

Fusarium infection0.8 %

Vibrio infection63.8 %

Unknown 2.1 %

Fusarium infection0.8 %

PRDV infection 80.2 %

1993

Diseases of kuruma prawn in Japan

Vibrio infection17.1 %

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Taura Syndrome Virus (TSV)-

- single-stranded RNA virus

- ‘cricket paralysis-like virus’, genus cripavirus

- non-enveloped icosahedron

- 32 nm

- 10,205 nucleotides (excluding 3’poly-A tail)

- Litopenaeus vannamei (principal host)

- US, Central and South America, Taiwan, Thailand

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(from AAHRI Newsletter)

reddish or pinkish coloration

blackish scar-like patchesTEM of TSV

(from Bonami et al., 1997)

-Exotic species should be kept in the closed system or in the limited area, not be released in the natural environment.

-Do not transfer or import the exotic shrimp without certification of virus-free stock.

-Do not co-culture the exotic species and endemic species.

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Yellowhead Disease Virus (YHDV)

Infectious Hypodermal and Haematopoietic Necrosis Virus (IHHNV)

(from Dr. Lightner’s Shrimp Disease Handbook)

Bent rostrums

Wide size range with numerous “runts”

Vibrio harveyi infection

Gill

Images of highly-sensitive camera

Body

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Detection Method

- PCR

- antibody-based technique

- in vitro virus replication

PCR

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Primer 2 Primer 3

Primer 1

Primer 4

643 bp

330 bp

WSSV partial genomic DNA (EcoR I- Hind III fragment)

889 bp

EcoRI HindIII

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Antibody-based technique

Procedure

ＴＣ

ＴＣ

1．Remove the swimming leg 2．Homogenize the leg

3．Add 150 µ l of buffer 4．Read the results in 15 min

PositiveNegative

5 min

from “Shrimple” of EnBioTec Laboratories, Japan

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Mechanism of Immunochromatograph

Positive

Negative

Test Line Control LineSample Loading

Anti-Rat IgG AntibodyGold Labeled Anti-WSSV Antibody(Rat IgG)

Immobilized Anti-WSSV Antibody (Rat IgG)

WSSV(Antigen)

from “Shrimple” of EnBioTec Laboratories, Japan

in vitro virus replication

- primary culture of lymphoid organ and gonad

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Rinse with KBS

Shrimp ovary（ 8 g）

Mince (1 ~ 2 mm3)

Digestion by enzyme

Filtration

Centrifugation （1,000 rpm, 5 min, 4°C）

Culture（25°C）

Cell culture procedure

Infected tissue

Homogenization

Filtration

Dilution

Inoculation Incubation

Normal

CPE

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Electron micrograph

5 days after the infection

PCR

Antibody-based technique (Shrimple)

- high sensitivity, high specificity, confirmatory diagnosis

- 3 - 6 hours

- expensive instruments & reagents

- training

- high sensitivity (~1-step PCR), high specificity, confirmatory diagnosis

- 30 minutes

- very practical (no training needed)

- rather expensive

in vitro culture- for research purpose

1 - 1.5 h

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Prevention of Viral Diseases

- Pond preparation, water quality management

- PCR checking (2-step PCR)

- Rinsing eggs

- Immunostimulant

- Viral Inhibitor

- Probiotics + “green water” technology

- SPF, SPR

PCR checking (2-step PCR)

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Table. Virus detection by 2-step PCR from wild-caught kuruma shrimp, Penaeus japonicus

Period Tissue

No. of positive /no. of shrimp examined

Pathogenicity study I*

Pathogenicity study II**

Spawner (71 - 140 g)

Apr - Sept 1996 Heart 51/203 (25.7 %) NT 10/10 (4 days)

Immature (13 - 48 g)

June - Dec 1996 Hemolymph 45/272 (16.5%) 28/28 (14 days)

10/10 (10 days)

* Wild-caught shrimp were kept in a small tank. ** Supernatant of heart homogenate was filtered and injected into cultured kuruma shrimp.

Table. Virus detection by 2-step PCR from wild-caught kuruma shrimp, Penaeus japonicus

Period Tissue

No. of positive /no. of shrimp examined

Pathogenicity study I*

Pathogenicity study II**

Spawner (71 - 140 g)

Apr - Sept 1996 Heart 51/203 (25.7 %) NT 10/10 (4 days)

Immature (13 - 48 g)

June - Dec 1996 Hemolymph 45/272 (16.5%) 28/28 (14 days)

10/10 (10 days)

* Wild-caught shrimp were kept in a small tank. ** Supernatant of heart homogenate was filtered and injected into cultured kuruma shrimp.

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Table. Application of 2-step PCR for detection of WSSV in shrimp through the culture period – hatchery

April 21 May 22 May 28 June 8-26

Spawners (200) Eggs Mysis Postlarvae Postlarvae - - - - - -

Rinsing with filtered seawater and iodine - -

+ destruction - - - -

+ (33.3%, 15/45)

non-treatment

- -

Stocked into 4 growout ponds

+: WSSV positive by 2-step PCR -: WSSV positive by 2-step PCR

Table. Application of 2-step PCR for detection of WSSV in shrimp through the culture period – growout ponds

Culture condition Growout ponds Rinsed

eggs Stocking density*

June 16 July 18 Aug. 1 Aug. 26 Sep. 25 Oct. 11

A Yes 30 - - - - - - B No 36 - - - - - - C No 31 - - - + (1/2) - D Mixed 100 - - +(3/5)# + (1/2) + (1/2) Water temperature (°C) 26 29 31 26 22 20 Average body weight (g) 0.12 1.9 3.8 6.0 17.8 19.4

+: WSSV positive by 2-step PCR -: WSSV positive by 2-step PCR *: pieces/m2 #: partially harvested to reduce the stocking density

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Rinsing eggs

Spawners

Aeration Aeration

Eggs and spawning material

SpawnedPCR

(receptaculumseminis)

Collection of eggs and debris

in the net (150-200 µm mesh)

Remove debris with 408 µm mesh

in the net

Iodine treatment (5 ppm 5 min)

from National Center for Stock Enhancement, with modification

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Immunostimulant

Survival of kuruma shrimp fed with peptidoglycan (PG) derived from Bifidobacterium thermophilum after challenge by WSSV

Days

Control

0.2 PG-i

0.2 PG-con

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80

Per c

ent

termination of PG feeding

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Viral Inhibitor

● Extracts of brown alga "Okinawa-mozuku", Cladosiphon okamuranus

PPF (partially purified fucoidan)

● Sulfated polysaccharide

● 133,000 ± 20,000

CH3

O

HO

ORO

nR = H or SO3

-

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Days after challenge

Fig. Survival rate of kuruma shrimp after WSSV challenge:

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9 10

100 mg

Control

10 mg

60 mg

20 mg

Surv

ival

rat

e (%

)

effective dose of PPF

Probiotics + “Green Water”Technology

Verschuere et al. (2000): a live microbialadjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of the ambient environment.

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(Next day)

Spread evenly on pond surface

Remove sludge on water surface

Probiotic + pond water (2-10 hrs)

Probiotics & “Green Water” Technology

(Corre et al., 1999)

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Summary

-- WSSV, TSV and other viral diseases are epizootic.

- Exotic species should not be introduced without pathogen-free certificate.

- Proper pond management and water quality management should be needed for sustainable shrimp culture.

- Gene analysis and the application to DNA array technology remain to be studied.

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Thank you very much for your kind attention

T. ITAMI, University of Miyazaki, Japan