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Departamento de Microbiología y ParasitologíaFacultad de Biología
Universidad de Santiago
The Use of Vaccines to Control Bacterial Infections in Marine Fish
J.L. Romalde
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GENERAL MEASURES OF PREVENTION
1) Adequate handling and maintenance of instalations
2) Control of sanitary quality of water and sediments
3) Control of food4) Control of imports and movements of fish and eggs5) Routinary control of health status of fish stocks6) Modification of physico-chemical parameters of
culture7) Control of wild ictiophage animals acting as
pathogen vectors
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Vaccination strategy
* Decision to which diseases to vaccinate against
* Vaccine type
* Vaccination method
* Timing of vaccination
* Revaccination
The chosen strategy needs to be easily integrated into the usual production protocols of the target fish species
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Before attempt vaccination:
* Determine maturity of immune system
# differences among fish species
salmonids mature at hatchingmarine fish 70-100 days post-hatching
Vaccination at short age in marine fish usually impliesone or more revaccinations
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Efficacy of vaccination:
* Type of vaccine formulationbacterins, live attenuated vaccines, DNA
* Monovalent vs. polyvalent vaccinesantigen competition?
* Route and strategy of administrationI.P., immersion (bath/dip), oral
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Route of administration
Intraperitoneal
Short Bath Long bath
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Route of administration
Intraperitoneal:
Water-based vaccines
Adjuvanted vaccines:
Mineral oil (side effects) 1st generation
Non-mineral oil* 2nd generation
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Determination of vaccine efficacy
RPS (Relative Percent Survival) estimation:
% mortality in vaccinated fishRPS = 1– x 100
% mortality in control fish
European Pharmacopeia:
RPS ≥ 70 for i.p. vaccinesRPS ≥ 60 for bath vaccines
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Streptococcosis:
* Streptococcus parauberisEndemic of turbotTemperatures higher than 15ºCAntigenically homogeneous
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* Commercial vaccinesVaccine ET-2 (USC/HIPRA Lab)Only effective when administered by I.P.
at 30 g fishLong-lasting protection (two years) with
RPS ≥ 80%.No booster required.Divalent formulation with T. maritimum also
available.
Streptococcosis:
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Vaccines developed and patented by theIctiopathology group of the Universidad de Santiago
* anti-V. anguillarum (GAVA-3) turbot, salmon* anti-P. piscicida (DI) seabream, seabass* anti-Streptococcus (ET-2) turbot* anti-F. maritimus (FM 95) turbot
transferred
Production&
Marketing
Laboratorios HIPRA S.A.
Gerona
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Biochemical and genetic characterization
Serological and antigenic studies
Characterizationof virulencefactors
Isolation of the pathogenin pure culture
Reisolation ofthe bacteria
Inoculation of healthy fish
Vaccine formulation
Evaluation of the efficacyAdministration route
Vaccination protocol
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Autovaccine preparationAutovaccine preparationInoculum
24 h
25ºC
48 h
25ºC
A=580 nm≥1
Formaldehide 1%
1 %
Shaking4 h
Toxicity test
Safety test Autovaccine
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Pseudomonas anguilliseptica
Two “O” serotypes
1 2 3
4 5 6 7
8 9 10
1 2 3
4 5 6 7
8 9 10
Representative strains of each serotype were selected for a divalent vaccine formulation
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Protection achieved by the pseusomonadiasis vaccine in turbot
Fish group Challenges
6 weeks 12 weeks
%M RPS %M RPS
I.P. 10 90 22 78
Control 100 100
Inoculated dose: 2.1 x 106 bacteria/fishWater temperature: 11 ± 1 ºCFish size at challenge: 6 g
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Vaccination field studies on gilthead seabreamM
orta
lity
obse
rved
February
March
Time
Non vaccinated fishVaccinated fish
Vaccination:October 2002
Water Tª:18 ± 2 ºC
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Tenacibaculum (Flexibacter) maritimumThe sole isolates, together with the seabream strains, constitute a “O” serogroup within this bacterial pathogen,clearly different to that comprised by the turbot isolates.
Strain PC-503.1 was then selected for the new vaccine formulation
Therefore, to develop a specific vaccine for sole is needed
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Protection yielded by the new flexibacteriosis vaccine for sole
Fish group Nº fish % Mortality RPS
I.P. 80 5 94.2
Control 80 85
Inoculated dose: 1 x 108 bacteria/fishWater temperature: 20 ± 1 ºCFish size at challenge: 50 gChallenge: 6 weeks after vaccination
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Acc
umul
ated
mor
talit
y
Days Days
Non vaccinated fishVaccinated fish
Divalent vaccine: Tenacibaculum maritimumPhotobacterium damselae subsp. piscicida
Monovalent vaccine: Photobacterium damselae subsp. piscicida
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• During 2005 and 2006: Isolation of E. tarda in turbot facilities associated with mortalities
Phenotypical and serological homogeneous
Some moleculardifferences (RAPD)
Turbotisolates
• Autovaccine: Isolate ACC 35.1 First isolate in
South Europa
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0
10
20
30
40
50
60
70
80
RPS
0 1 2 3
Months after vaccination
RPS in vaccinated turbot (10 gr)
IP BATH
Challenge
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Level of antibodies in 10 g turbot IP vaccinated
0
0,5
1
1,5
2
2,5
Level ofantibodies
0 1 2 3
Months after vaccination
ELISA
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Divalent / trivalent vaccines
00,5
11,5
22,5
Level of Antibodies
S.parauberis
E. tarda T.maritimum
12
Level of antibodies one month after vaccination
01020304050607080
RPS
0 1 3Months after vaccination
RPS for E. tarda infection in turbot
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AREAS OF FUTURE DEVELOPMENT IN FISH VACCINATION
* To establish a vaccination program adequate for each fish species
* Development and optimization of polyvalent vaccines * To develop and improve the oral vaccination protocols* To evaluate non-mineral oil adjuvanted bacterins and
microencapsulated vaccines
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Thanks for your attention
Grazie per l’attenzione