RECENT DEVELOPMENTS IN

VACCINATION IN FISH

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• Mr. MAHIDA PARVEZ JILANI

• Department of Aquaculture

BACKGROUND - AQACULTURE

• Agriculture and fisheries will soon reach the maximum capacity in global food production

• Consequently, food from aquaculture will be more important for future food security. Aquaculture must be sustainable with minimal use of antibiotics and chemicals

• Correct use of vaccines will contribute to sustainability in aquaculture

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BACK GROUND - VACCINE

The first vaccines for aquaculture were simple products like formalin inactivated bacterial cultures

Most modern fish vaccines are research based products The way from research to licensed vaccines is long and

expensive A licensed vaccine may be required for import of

aquaculture products Many countries (EU-countries and USA) and organizations (FAO, OIE, WTO) have strict import requirements related to the use of vaccines in aquaculture production03/05/15 3

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• The first vaccines against Fish infectious bacterial

diseases were developed in 1970.

• It is commercial in 1980.

• This has contributed significantly to the growth of

the industry to consumer acceptance of farm raised.

• Research over many year very few Anti-viral

vaccines are available.

• There are no commercial vaccines against parasite.

HISTORY OF VACCINES

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Aquaculture Vaccines - History

• Furunculosis - Proof of concept - Duff (1942)

• Commercial bacterial vaccines (mid 1970’s)

• Oil adjuvanted injectable vaccines (early 1990’s)

• Commercial viral vaccines (mid 1990’s)

• Commercial recombinant vaccines (late 1990’s)

• Commercial DNA vaccine (2005)

• Reverse engineered & multiepitope vaccines (2007)

Edward Jenner (1749-1823)

• Edward Jenner, for the first time in 1796 used cow pox vaccine against the small pox

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Louis Pasteur (1822-1895)

• Introduced the term‘virus-vaccin’ synonymous with attenuated microbe

• Germ theory of disease rabies vaccine in 1885

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From Louis Pasteur to fish vaccinology

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vaccines

• “Vaccines are preparations of antigens derived from pathogenic organisms, rendered non-pathogenic by various means, which will stimulate the immune system in such a way as to increase the resistance to disease from subsequent infection by a pathogen.”

A.E. Ellis, 1988

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The concept of vaccination

Vaccination

Pathogens (bacteria, viruses…⃝

Resistance to infection

Mortality

Treatment

Healthy

Infection

Diseased

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Principle of vaccination

• The major principle of vaccination is memory and specificity”

• Immunological Memory: The ability of the immune system, upon interaction with a previously encountered antigen produce a stronger and faster immune response

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Specificity

• The exquisite specificity of the immune system allows it to selectively recognize billions of different foreign antigens, while maintaining tolerance to an equally diverse panel of self antigens

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Development of Fish VaccinesDevelopment of Fish VaccinesDevelopment of Fish VaccinesDevelopment of Fish Vaccines

Disease InformationDisease InformationDisease InformationDisease Information

A etiology of DiseaseA etiology of DiseaseA etiology of DiseaseA etiology of Disease

Characterization of IsolatesCharacterization of IsolatesCharacterization of IsolatesCharacterization of Isolates

Definition of DiseaseDefinition of DiseaseDefinition of DiseaseDefinition of Disease

Challenge Model Devel.Challenge Model Devel.Challenge Model Devel.Challenge Model Devel.

Vaccine DevelopmentVaccine DevelopmentVaccine DevelopmentVaccine Development

Vaccine ProductionVaccine Production Vaccine ProductionVaccine Production

Lab Vaccination TestsLab Vaccination TestsLab Vaccination TestsLab Vaccination Tests

Pre-Licensing StudiesPre-Licensing StudiesPre-Licensing StudiesPre-Licensing Studies

LicensingLicensingLicensingLicensing

Marketing PreparationMarketing PreparationMarketing PreparationMarketing Preparation

Product IntroductionProduct IntroductionProduct IntroductionProduct Introduction

Normally 5 – 8 Normally 5 – 8 years in R&Dyears in R&D

Normally 5 – 8 Normally 5 – 8 years in R&Dyears in R&D

Epidemiology and disease Epidemiology and disease investigationinvestigation

Laboratory phaseLaboratory phase

Field and licencing Field and licencing phasephase

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Important considerations for fishvaccination

• Species

• Status of the immune system

• Production cycle and life history

• What diseases do you want to control?

• When do these diseases occur?

• Farming technology (Handling, mechanization)

• Environment (temperature, salinity)

• Stress factors, nutrition and cost benefit03/05/15 15

Globally Important Bacterial Pathogens in Aquaculture

• Listonella

• Vibrio anguillarum

• Aeromonas salmonicida

• Yersinia ruckeri

• Renibacterium salmonarium

• Lactococcus/Streptococcus sp

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Globally Important Viral Pathogens in Aquaculture

Infectious Pancreatic Necrosis (IPN)Salmon Pancreas Disease (SPD)Sleeping Disease of Trout (SD)Infectious Salmon Anaemia(ISA)Viral Nervous Necrosis (VNN)Infectious Haematopoietic Necrosis (IHN)

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The Ideal characteristics of Fish Vaccine ?

• Sustained immunity and protection

• Early mass application

• Efficacious for a broad number of species

• Safe

• Cheap and cost effective

• Easily produced

• Stable

• Will not interfere with diagnosis

• Easily licensed03/05/15 18

What Fish vaccines are Used?

• Most commonly used antigens in fish vaccines to date are inactivated or killed bacterial and viral• Most successful use has been against

Furunculosis (Aeromonas salmonicida) in salmon• Several inactivated antigens now present in

most commonly used salmon vaccines, i.e. multivalent

• Recombinant sub unit vaccine used for IPN• DNA vaccines for IHN and VHS in development

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• Immune system of fish Similarities and differences compared with warm blooded animals

• Innate immunity is more important in fish

Specific immunity is less developed in fish

• Maternal immunity cannot be used for disease prevention in fish

• Protective immunity can be developed even in young fish

• Salmonid fish at a size of less than 5 grams develop immunity Variation between fish species

• Salmonid fish differ from cod fish

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TYPES OF VACCINES

1) INACTIVATED VACCINES•Most bacterial vaccines used in aquaculture to date have been inactivated vaccines obtained from broth culture of a specific strain(s) subjected to subsequent formalin inactivation.

•The best results are obtained with those bacterins that include both bacterins cells and extracellular products.

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• Whereas with some vaccines acceptable levels of protection are achieved with aqueous formulation administered by injection or immersion.

• Bacterins such as those devised for Salmonids against Aeromonas salmonicidia an accepatable level of protection can only be achieved by immuunization with oil –adjuvanted bacterins delivered by injection.

2) LIVE VACCINES

These should have many advantages in Aquaculture.

Live vaccines also have the advantage that stimulate the cellular branch of the immune system.

Some live vaccines have been tested experimentally are Aermonas salmonicida, Edwardiella tarda E.ictaluri.

At present ,only on E.ictaluri attenuated live vaccine has licensed in then USA to be used by both in 9-days old fish to prevent ESC of cat fish.

Cont…

3) DNA vaccines

•DNA vaccines have theoretical advantage over conventional vaccines in mammals the specific immune response after DNA vaccination encompasses antibodies ,T-helper cells and cytotoxic cells.

•As the DNA sequence encodes only a single microbial gene , there should be no possibility of reversion to virulence.

•It is a critical factor to factor in enviromental saftey in Aquaculture.

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4) BIOFILM VACCINESBacterial biofilm is a colony of high density of cell embedded in a glycocalyx matrix on a substrate, which has been demonstrated to be resistant to action of antibiotics chemicals and host immune system. Bacteria biofilm on suitable substrate after inactivation can be used as a successful oral vaccine.

5) RECOMBINANT PROTEIN VACCINES It starts with identification of immunogenic subunit or protein from a pathogen of interest followed by the genes involved in coding for them which can be introduced into a vector, over expressed in expression hosts and can be used as recombinant protein vaccines .

Adjuvants - a challenge• Adjuvants are necessary in order to achieve

acceptable protection against some diseases

• Oil adjuvants may cause adherence in peritoneal cavity and muscle pigmentation

• The side effects caused by some adjuvants are not acceptable from an animal welfare point of view

• The search for improved adjuvants and factors contributing to the side effects is a great challenge

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VACCINATION METHODS

Direct immersion

Oral

Injection

Vaccination Methods

Injection vaccination–Intraperitoneal (adjuvant or not)–Intramuscular (DNA )

Immersion vaccination–Bath method–Dip method–Spray vaccination

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Vaccination MethodsSpray vaccination:

A variant of immersion could be used

in larger fish where injection not possible

Oral vaccination:Micro-encapsulation methodBio-encapsulation method

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Fish vaccination process• Fish are transported in pipes from the

rearing tanks to an anesthetic bath.

• The anaesthetized fish are injected by the vaccination team.

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Injection vaccination -by hand

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Injection by machine

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Fish immersion vaccination

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Oral vaccination

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Advantages & disadvantages ofimmersion vaccination

Suitable for mass vaccination of all sizes of fish Reduced stress for fish Lower labour costs Less risk to vaccination team

• Major disadvantages are the large amount of vaccine required and lower level of protection and duration of immunity

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Advantages & disadvantages ofInjection vaccination

• Most common method of vaccine delivery in fish

• Highly efficient in generating both humoral (antibody) and cellular cytotoxic response

• Disadvantages : Unsuitable for small fish Needs sophisticated machinery or large skilled

workforce Significant handling stress and risk of post

vaccination fungal infections Local reactions

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Advantages & disadvantages ofOral vaccination

Vaccine mixed with feed Easiest method for mass vaccination of all sizes

of fish Saves labour and avoids stress

• Disadvantages :Large quantities of antigen required Requires all fish to be feeding Protection generally weak and of short duration

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Choice of Application method

Determined by Vaccination window Size of fish Duration of protection required Type of pathogen Type of immune reaction Single versus multivalent product Cost of vaccine

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The ideal vaccination strategy• Oral vaccines of high efficacy which could be given

in or on the food as required given required

• Immersion vaccine injection oral boosters as

required

• Currently salmonid industry relies on a single

industry injection of a multivalent vaccine

• Danger of antigen overload

• New vaccines and vaccine strategy required

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Successful vaccination of fish

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Successful vaccination against bacterialdiseases

• Atlantic salmon and rainbow vibriosis trout are vaccinated against:

• vibriosis (Vibrio anguillarum)• cold water vibriosis (Vibrio salmonicida),

• Furunculosis (Aeromonas salmonicida) prior to sea transfer with an injectable adjuvanted vaccine

(SOURCE : Håstein et al. 2005 Dev.Biol 121, 55-74)

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Viral vaccines

Inactivated vaccines are used against infectious pancreatic necrosis (IPN), infectious salmon anaemia (ISA) and pancreas disease (PD) with some effectA DNA vaccine against infectious haematopoietic necrosis (IHN) is licenced in Canada. The vaccine gives acceptable protection…….

Research on DNA-vaccines for several diseases (IPN,VHS, koi herpes virus-infection, spring viraemia in carp)

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New technologies

• Sub-unit vaccines

• Deletion mutants

• Live vectored vaccines

• DNA-vaccines

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Subunit vaccines

• Subunit vaccines contain a portion of the infectious agent which is essential for stimulation of protective immunity• Subunit fish vaccines are commercially

available• Example: IPN-vaccine for salmon fish• Advantage: Safe and inexpensive• Disadvantage: No intracellular replication

and inadequate cellular immunity

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Deletion mutant

• No deletion mutant fish vaccines are commercially available

• Efficacy: Mimic pathogens and stimulate mucosal immunity and cell-mediated immunity

• Safety: Reversion to virulence less likely in genetically engineered vaccines

• Can be used as marker or DIVA-vaccines (differentiation infected from vaccinated)

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Live vectored vaccines

Efficacy: can be used for viruses with no or poor replication

Cellular immunity is stimulated with the right vector

Safety: Safe, if the vector is non-pathogenic Several live vectored vaccines for terrestrial

animals, but none for fish

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DNA-vaccines

•Efficacy: A DNA-vaccine against IHN is licenced in Canada

•The vaccine gives acceptable protection

•Safety: Fish vaccinated with DNA-vaccines are considered to be gene modified organisms (GMO) in some countries

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Future prospectus:-

To achieve progress in fish vaccinology, an increase in the co-operation between basic and applied science is needed.

Improvement in oral immunity with biodegradable microparticle based vaccines to be used for booster vaccination.

Development of new non-mineral oil adjuvants lacking side effects.

Development of poly valent vaccines and stabilization of a vaccine calendar appropriate each economically important fish species.

Investigation of the mechanisms of immunoglobulin transfer from pre spanning females to offsprings as a useful way of protecting fish against pathogens which affects early life stages.

SUMMARY

Vaccination of aquatic animals is a basis for good health, good economy and sustainability

Efficacious vaccines are available for many diseases Main challenges for future development are● live vaccines● Oral vaccines● Vaccines for virus and intracellular bacteria● Effective adjuvants with minimal side-effects Molecular biology is a useful tool for research,

production and control of new fish vaccines

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We make aquaculture progress

Thank you

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