Departamento de Microbiología y ParasitologíaFacultad de Biología

Universidad de Santiago

The Use of Vaccines to Control Bacterial Infections in Marine Fish

J.L. Romalde

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

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

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

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

Route of administration

Intraperitoneal

Short Bath Long bath

Route of administration

Intraperitoneal:

Water-based vaccines

Adjuvanted vaccines:

Mineral oil (side effects) 1st generation

Non-mineral oil* 2nd generation

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

Streptococcosis:

* Streptococcus parauberisEndemic of turbotTemperatures higher than 15ºCAntigenically homogeneous

* 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:

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

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

Autovaccine preparationAutovaccine preparationInoculum

24 h

25ºC

48 h

25ºC

A=580 nm≥1

Formaldehide 1%

1 %

Shaking4 h

Toxicity test

Safety test Autovaccine

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

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

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

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

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

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

• 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

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

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

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

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

Thanks for your attention

Grazie per l’attenzione