Download Pathology Division, NFRDI

YSLME
17th June 2009
RMC
Status of Fish Vaccine Development in Korea
Myoung Ae PARK
Pathology Division
National Fisheries Research and Development Institute
Contents
Status of fish disease in Korea
Developed fish vaccine in Korea
Research trends of fish vaccine
Pathology Division, NFRDI
Aquaculture Production in Korea (2007)
Production of Marine Aquaculture
Fish: 97,663 M/T (7%)
Shell fish: 478,646 M/T
Seaweed: 792,953 M/T(60%)
Crustacea: 1,321 M/T
Other : 15,221 M/T
Production of Inland Aquaculture
Fish: 23,424 M/T
Shell fish: 1,813 M/T
Crustacea: 177 M/T
Other: 132 M/T
Data: MIFFAF
Pathology Division, NFRDI
Aquaculture Production in Korea (2007)
Production of Marine Cultured Fish
M/T
Red
Others
seabream 12%
7%
Sea bass
3%
Rockfish
36%
Olive
flounder
42%
Total
97,663
Olive flounder
41,171
Rock fish
35,564
Red seabream
7,213
Sea bass
2,361
Other
11,354
Data: MIFFAF
Pathology Division, NFRDI
Status of fish disease in Korea
Fish Disease Pattern in Korea
2005
bacteria
bacteria+bacteria
bacteria+parasite
2000
parasite
virus
others
1996
0%
20%
40%
60%
80%
100%
In the 1990’s, losses due to disease were about 5% but this has now
increased to about 20%. Major infection causes change from bacterial
problems to complex problems.
Pathology Division, NFRDI
Status of fish disease in Korea
Major infectious diseases of marine cultured fish
Disease
Causative agent
Affected species
Red seabream iridoviral disease (RSIVD)
Iridovirus (RSIV)
Red sea bream,
Rock bream, flounder
Viral nervous necrosis (VNN)
Nodavirus (VNNV)
Several fishes
Viral hemorrhagic septicemia (VHS) virus
infection
VHSV
Olive flounder
Vibriosis
Olive flounder
Streptococosis
Vibrio harveyi,
V. ichthyoenteri
Streptococcus iniae,
S. parauberis
Edwardsiellosis
Edwardsiella tarda
Olive flounder
Scuticociellosis
Uronema marinum
Olive flounder
White spot disease
Cryptocaryon irritans
Olive flounder
Olive flounder
Pathology Division, NFRDI
Necessity of vaccine development
Disease Control



Rapid detection of pathogens and control by antibiotics
Good husbandry
Prevention of disease by vaccination
Vaccines






Major area for growth in aquaculture
Reduce the need for antibiotics and chemicals
Save costs
Reduce problems with antibiotic resistance
Reduce concerns over residue levels and environmental impacts
Control significant diseases
Pathology Division, NFRDI
Types of Vaccine

Inactivated whole cell

Adjuvanted

Sub-unit

Recombinant

Live attenuated

Synthetic (peptide)

DNA vaccines
Development
cost
The first commercial vaccine were licenced in the USA in 1970’s
against enteric redmouth disease, vibriosis and furunculosis
Pathology Division, NFRDI
Liscenced Vaccines for Fish
Enteric Redmouth (ERM)
Vibrio anguillarum
Furunculosis
Vibrio salmonicida
Vibriosis/Furunculosis
Vibriosis/Furunculosis/Coldwater Vibri
osis/Moritella viscosa
Vibriosis/Furunculosis/Coldwater Vibri
osis/Moritella viscosa/ IPN Virus
Vibriosis for cod
IPN Virus
ISA Virus
Warmwater Vibrio spp
Pasteurella
Pasteurella/Vibriosis
SVC virus Lactococcus garvieae/Strep
tococcus iniae
Koi Herpes Virus
Aeromonas hydrophila
Carp Erythrodermatitis/Ulcer disease
Piscirickettsia salmonis
Flavobacterium psychrophilum
Nodavirus
Pancreas Disease Virus
Edwardsiella ictaluri
Pathology Division, NFRDI
Status of Fish Vaccine Development
Developed Vaccines for Fish in
Korea
Vaccine
Fish
Method
Status
Inactivated
Edwardsiella
Flounder
Immersion
Commercially available
Inactivated Iridovirus
Sea breams
Injection
Technique was transferred into
commercial co. in 2002
Inactivated
Streptococcus
Flounder
Injection
Commercially available
Inactivated Strepto
+Edward
Flounder
Injection
Under developing
Recombinant
Iridovirus
Sea breams
Flounder
Injection
Technique was transferred into
commercial co. in 2006
Pathology Division, NFRDI
Red sea bream Iridoviral Disease
Red sea bream Iridoviral Disease (RSIVD)

Over 30 cultured and wild fish hosts including:
Red sea bream, olive flounder, Sea bream, Barramundi, Japanese sea bass,
Largemouth bass, Northern bluefin tuna,
Several grouper species, Golden striped amberjack
2005

N
38o
Regions affected include:
Japan, Korea, Taiwan, Singapore

Diagnosis
Has been isolated in cell culture.
PCR commonly used for direct identification
or confirmation. FAT used for direct identification

Control
Cheonsuman
Pohang
36o
Ulsan
Geoje
Yeosu
Busan
Yeonggwang
Tongyeong
Wando
34o
Jeju
126o
Inactivated vaccine commercially available
DNA vaccines have been developed and are being evaluated
128o
130oE
Pathology Division, NFRDI
Red sea bream Iridoviral Disease
Red sea bream Iridoviral Disease (RSIVD)





Causes low to high mortality.
Highly infectious.
Affects a wide range of food fish.
Severe anaemia, haemorrhages in gills,
enlarged spleen.
Histology: enlarged cells
Pathology Division, NFRDI
Red sea bream Iridoviral Disease
Spread to Other Species
Iridovirus
Olive flounder
Red seabream
Parrot fish
Rockfish
Sea bass
Pathology Division, NFRDI
Red sea bream Iridoviral Disease
Process of Inactivated Iridovirus Vaccine
CPE
Added formalin
after harvest the sup
Harvest of cell
and determined TCID50
Remove formalin
using PBS
Vaccine
Pathology Division, NFRDI
Red sea bream Iridoviral Disease
Cumulative Mortality of Iridovirus Vaccine by Concentration
100
Non vaccination
108TCID50/fish infection
80
104TCID50/fish infection
Tissue Culture Infectious Dose
60
40
20
0
3
6
9
12
15
18
21
24
Day
Inactivated iridovirus vaccine was injected to parrot fish,
Challenged with live iridovirus
Pathology Division, NFRDI
Red sea bream Iridoviral Disease
Cumulative Mortality of 2nd Vaccination of Iridovirus
100
Non vaccination
108TCID50/fish infection
80
104TCID50/fish infection
Tissue Culture Infectious Dose
60
40
20
0
3
6
9
12
15
18
21
24
Day
Pathology Division, NFRDI
Red sea bream Iridoviral Disease
DNA Vaccine & Recombinant Vaccine
Virus nucleotide
Virus protein
Transformation to
cells
Vector
DNA vaccine
Isolation of virus
Expression of
nucleotide
protein
Recombinant
vaccine
mortality
Antibody titer
Pathology Division, NFRDI
Red sea bream Iridoviral Disease
The Problems of Virus Vaccine Development
Selection of host cell line
Purification of virus
Safety issues of genetically modified organisms
Decision of the time of vaccination
Pathology Division, NFRDI
Plans of Virus Vaccine Development
VHSV: Olive flounder
Nodavirus: Olive flounder, Grouper, Sea bass
KHV: Colored carp
SVC: Carp
Pathology Division, NFRDI
Edwardsiellosis
 Pathogen: Edwardsiella tarda
 Target organism:
Flounder, red sea bream
 Outbreak period:
All the year round
Caused by chronic
High cumulative mortality
 Symptom:
Expanded abdomen, rupture,
Darkness, ascites, exophthalmia
Pathology Division, NFRDI
Edwardsiellosis
Efficacy of the Immune System to Vaccination
Agglut in a t ion t it er ( log2 )
5
control
4
once immun.
twice immun.
3
2
1
0
1s t
immun.
1
2nd
immun.
3
4
5
6
7
8
Weeks after immunization
Agglutination titer after vaccination
Pathology Division, NFRDI
Edwardsiellosis
추가접종(Booster) 결과
Cumulative mortality of vaccinated olive flounder
infected with Edwardsiella tarda
Vaccination
Number of
fish
Immunized
and challenged
1st
10
10
10
10
Dose of challenged
(cells/fish)
Number of
fish died
RPS (%)
2
5
8
9
50
17
11
10
2nd*
10
10
10
10
7.1×104
7.1×105
7.1×106
7.1×107
7.1×104
7.1×105
7.1×106
7.1×107
1
1
3
5
75
83
67
50
Control**
10
10
10
10
7.1×104
7.1×105
7.1×106
7.1×107
4
6
9
10
* Second immunization was practiced at 2 weeks after first immunization
** Unimmunized
Pathology Division, NFRDI
Edwardsiellosis
Mortality of olive flounder with after challenged virulent
Edwardsiella tarda at week 4 after immunization by fish size
Fish size
(body weight)
Number of fish immunized
and challenged
Dose of challenged
(cells/fish)
Number of fish died
Mortality (%)
1.2g
10
10
10
10
6.9×104
6.9×105
6.9×106
6.9×107
2
4
6
9
20
40
60
90
6.5g
10
10
10
10
6.9×104
6.9×105
6.9×106
6.9×107
3
3
7
8
30
30
70
80
Pathology Division, NFRDI
Edwardsiellosis
Efficacy of immersion vaccination against
Edwardsiella tarda on the cultured olive flounder
Number of
tested fish
Vaccinatio
n
Control
5,000*
5,000
Monthly mortality
4
5
6
7
8
9
10
11
5
10
7
5
0
2
7
36
19
643
15
318
0
75
10
38
Cumulative RPS
mortality
(%)
63
1,127
94.4
* Average body weight : 1.2g (4∼5㎝)
Pathology Division, NFRDI
Streptococcosis
• Pathogen: Streptococcus iniae,
S. parauberis,
Lactococcus garvieae
• Target species: Flounder, Rock fish,
Yellow tail, Red sea bream
• Outbreak: High temp. season (July-Nov)
• Symptom:
Haemorrhages within the opecular cavity,
abdomen and internal organs,
ascites, enlargement of the spleen,
bloody spot inside of the opercula and
peritoneum
Pathology Division, NFRDI
Streptococcosis
Maintain stability and high defense
preserved after 15 months
250
Prime injection
agglutination titer
200
Boost injection
150
100
50
0
2
4
8
12
w eeks after vaccination
Change of agglutination titer after 15months
Pathology Division, NFRDI
Mixed Vaccine
(Edwardsiellosis+Streptococcosis)
Edwardsiella tarda
Streptococcus iniae
Flounder,
Gram negative
Gram positive
High temperature
The onset of the disease / single infection, mixed infection
Pathology Division, NFRDI
Fish Bacteria Vaccine
Selection of Bacterial Strains for Mixed Vaccine
Characterization
of pathogen
Biochemical and
genetic
characterization
Isolation of the pathogen in pure culture
Serological and
antigenic studies
Charactrization
of virulence
factors
Evaluation of the efficacy
Vaccine formulation
Pathology Division, NFRDI
Fish Bacteria Vaccine
Efficacy of Immune Induction Using Mixed Vaccine
Index of efficacy
Ab titeration
Vaccination
Serum separation
Cumulative
mortality
Formation of antibodies to pathogens
Pathology Division, NFRDI
Mixed Vaccine
(Edwardsiellosis+Streptococcosis)
Validation of the Superior Efficacy of Bacteria Mixed Vaccine
the protection of two kinds for the disease is acquired just
one time vaccination
Relative survival rate of mixed vaccine is higher than that
of single vaccine in 9-21%
Immune effects of a short period of time is expected without
additional inoculation
(A)
(B)
100
SE10
S10
PS
% Cumulative mortality
% Cumulative mortality
100
80
60
40
20
SE10
E10
PS
80
60
40
20
0
0
0
2
4
6
8
10
Days after challenge
12
14
0
2
4
6
8
10
12
14
Days after challenge
연쇄+에드와드 혼합백신의 단독 및 혼합투여 후 S. iniae 와 E. tarda 에 대한 누적폐사율
Pathology Division, NFRDI
History and Vision of Fish Vaccine
2020
2020
Vaccine program
2015
Oil-based vaccine
2010
Polyvalent vaccine(E.tarda+S.parauberis+V.harveyi
2009
2007
Mixed vaccine(Edwardsiella tarda + Streptococcus iniae)
2005
Recombinant vaccine(Iridovirus)
Injection vaccine(Streptococcus iniae)
2004
Immersion vaccine(Edwardsiella tarda)
2000
Pathology Division, NFRDI
Research Trends in Korea
Future
Present
Treatment by antibiotics
Focus on prevention
[ problems ]
• Increased antibiotics resistance bacteria
• Food safety threats by residual antibiotics
• Mixed infection
• Viral disease
 Highly efficacy vaccine development
 Development of immunostimulants
 Standardization of diagnostic methods
 Development of disinfection protocols
Eat fish, live longer !!!
Eat clam, last longer !!!
Eat oyster, love longer !!!
Pathology Division, NFRDI
Thank you for your attention
NFRDI