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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: [email protected]
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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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