Vaccine

Prevention of disease through vaccination is an important approach

to maintain fish health.

Exposure of a fish to a pathogen stimulates the immune response

and the fish will develop the memory to these pathogens so that it
can combat the pathogen very quickly during future encounter.

This adaptive immunity is specific against the pathogen and persists

for a relatively long period.

This persistence is because of an adaptive change in the population

of lymphocyte which results from the exposure of the fish to the
pathogen that consist the antigen.

Vaccines exploit the two key elements of adaptive immunity and

those are specificity and memory.
This is because after vaccination the adaptive immune
system responds specifically to the invading pathogen and
the response become stronger and stronger on the second
and subsequent exposure to the pathogen.

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.
Inducing a protective immune response to a pathogenic organism
before the individual becomes naturally exposed to it seems at first
sight is a highly sensible way of preventing an infectious disease.

Unfortunately, achieving this goal not an easy task.

Vaccines must be safe (no side effects) and potent (induce a high level
of protection) and although occasionally it has been possible to
achieve these characteristics using simple procedures (usually heat or
chemical inactivation of cultures of pathogenic microorganisms)

In most cases, some degree of antigen purification or enrichment is

necessary and the problem lies in identifying the relevant antigens, out
of many hundreds present, which are important in stimulating a
protective immune response.
Vaccines are generally of two types:
Dead vaccines, which are composed of inactivated pathogens or
extracts
Live vaccines, which are attenuated pathogens with no or low
virulence."
Dead vaccines
Inactivated virus or bacterial antigens
Sub-unit vaccines
Recombinant vaccines
Live vaccines
Attenuated live vaccine
Gene deleted live vaccine

The Ideal Fish Vaccine should have

Sustained immunity and protection
Early mass application
Efficacious for a broad number of species
Safe to use
Cheaply available
Easily produced
Should have stable antigen
Can be stored for long time.
Will not interfere with diagnosis
Easily licensed
Types of vaccine:
Killed whole cell vaccines: Killed whole cell vaccine is a
suspension of heat or chemical-killed pathogens that are able to
induce specific protective immune response against those
pathogens when administered into the host.

Killed whole cell vaccines are administrated when it is unsafe to

use live microorganisms. These vaccines are prepared from
normal wild type bacteria that are made nonpathogenic usually
by treating with heat, formaldehyde or gamma irradiation.

Killed whole cell vaccine can be used in controlling some of the

important fish bacterial pathogens like V. Anguiliarum, V. ordalli,
Y. ruckeri, E. tarda and A. salmonicida.

Among carp virus killed vaccine of spring viremia of carp virus

(SVCV) was commercially available for some years.
Advantages:
•Safe to use
•Cheaper than live attenuated vaccine
•Storage is easier than live vaccine
Disadvantages:
•Large number of microorganisms is required to stimulate immunity.
•High cost of their production in cell culture,
•Difficulty in the purification and delivery.
•In general killed vaccines alone trigger only the humoral immune
response
•Needs to be given booster doses
•Presence of some un-inactivated microbes can lead to vaccine-
associated disease.
•Inactivation, by formaldehyde may alter antigenicity.
Live-attenuated vaccines: Live-attenuated vaccine is a suspension
of attenuated live pathogens that are able to replicate inside the
host and induce protective immune response but unable to cause
disease.

To make an attenuated vaccine, the pathogen is grown in foreign

host such as animals, embryonated eggs or in tissue culture under
controlled conditions and altered to a non-pathogenic form to
make it less virulent.

They mimic the actual infection by pathogens and hence a small

dose of vaccine is enough to induce long lasting protective immune
response.

Live attenuated vaccines can induce both humoral and cell-

mediated immune responses.
Advantages
•Live-attenuated vaccine can stimulate cellular immune responses,
memory cells as well as humoral immune responses.
•Administrating lesser quantities is sufficient to induce protection.
•Single administrating of vaccine is sufficient to produce high
efficacy and long lived immunity.
•Administration of live attenuated vaccine has a possibility to
stimulate immune response to all protective antigens.

Disadvantages
•Live-attenuated vaccine may rarely revert to its virulence form and
cause disease.
•Live-attenuated vaccine cannot be given to immunosuppressed
individuals.
•Since they are live they require proper storage.
Subunit Vaccines
Subunit vaccines are the purified antigens of the whole organisms.

These antigens are capable of stimulating antibody response.

Subunit vaccines may contain toxoids, subcellular fragments, or

surface antigens. The effect of subunit vaccine can be increased by
administrating these subunits with adjuvants.
Advantages
•They can be safely administrated to the immunosuppressed
animal.
•The side effects caused by sub unit vaccines are very rare.
Disadvantages
•Since the vaccines are purified, the purification process may
affect antigen in retaining its native form, so the organism may not
recognize the antigen on pathogen.
•These proteins may not stimulate the immune response.
Recombinant vaccines:
DNA vaccine: This vaccine is still in experimental form. Here the
gene of the pathogen is clone into vector and the vector is inserted
into the animal.
Once the vector is introduced, the protein that act as antigen is
produced inside the host and stimulate the immune response. DNA
vaccine can induce both humoral and cellular immunity.
 Advantages
•DNA is stable, hence can be easily stored and transported.
•DNA sequence can be changed easily in the laboratory.
•DNA does not replicate and encodes only the proteins of interest
Disadvantages:
•Insertional mutation may be possible
•Anti DNA antibodies may be produced against the inserted vector
•Since the antigen is expressed in the host immunologic tolerance
may develop.
•Ethical issues.
Vaccination Methods:
Vaccines may be delivered to fish by
Intraperitoneal or intramuscular injection method
Oral vaccine method
Direct immersion method

Vaccination is done to the healthy fish because it is a preventive

measure and not a cure.
Oral Vaccination: During the oral vaccination, the vaccine is given
orally and it may be either through feed, where it is either mixed
along with feed or coated on top of the feed or bio-encapsulated.

During oral vaccination proper care must be taken to ensure that

the antigen is successfully absorbed into the body.

It is because the antigens incorporated in feed may be the heat

sensitivity or the digestive system may digest the antigen.
So when the vaccine is coated on the feed usually a coating agent
is applied to prevent leaching of vaccine or to prevent the
digestion of an antigen in the acidic environment of the fish
stomach.

Bio encapsulations are used usually when the fish fry are to be
vaccinated, Bio encapsulation include the live feed such as
Artemia nauplii, copepods or rotifers which are fed after
incubation in a vaccine suspension.

During incubation they accumulate the antigen in their digestive

tract and as such, transform themselves into living microcapsules.
Advantages
•Vaccine mixed with feed
•Easiest method for mass vaccination of all sizes of fish
•Saves labour
•Avoids stress to fish

Disadvantages
•Large quantities of vaccines are required
•Some fish may not consume the feed.
•Protection generally weak and of short duration
Immersion vaccination: Fish being aquatic in nature it is
constantly surrounded by the microorganisms of which some
may be pathogenic ones.

These fish have a mechanism to recognize these pathogens and

elicit an immune response.

So when the fish comes in contact with vaccine during immersion

these vaccine is absorbed by the skin and gills, the specialized
cells such as antibody-secreting cells are activated and will
protect the fish during the later stage of exposure to the
pathogen.
Immersion vaccine can be applied in two ways:-
Dip method
Bath method
Dip method: In dip vaccination method fishes are immersed in a
highly concentrated vaccine solution for very short time say usually
few minutes.
Bath method: In bath vaccination method fishes are immersed in a
lower concentration of vaccine solution for a longer period, usually
one to several hours.
Advantages
• Suitable for mass vaccination of all sizes of fish
• Reduced stress for fish
• Lower labour costs
• Less risk to vaccination team
Disadvantages
• Major disadvantages are the large amount of vaccine required
• Lower level of protection
• Low duration of immunity
Injection vaccination: Vaccination by injection works well with
fish that are 5–10 g or larger and the injection vaccination is very
popular in salmon industry.

Light anaesthesia is given to the fish before injection to prevent

mechanical injuries and also to overcome handling stress.

Vaccine can be administrated through intramuscular or

intraperitonial with intraperitonial vaccination is beeing very
common.

The immune response development depends on the needle and

method of injection.

Short needles might deliver the vaccine in the muscle and cause
inflammation and a bad immune response, while long needles
may damage the internal organ.
The right thickness of needle is also important because the
viscous vaccine has to pass through the needle and if the needle
is too large the vaccine may fall back and may lead to infection.

Once vaccination is administrated it will take weeks to develop

immune response, so it is important maintain the fish in ambient
temperature and not to stress fish because temperature variation
and stress may suppress the immune system.
Advantages
•Provide protection for long duration
•multiple antigens can be administrated in single injection
•every fish in farm can be vaccinated
•every fish in farm receives the correct dose
•Highly efficient in generating both humoral (antibody) and
cellular cytotoxic response

Disadvantages
•Needs sophisticated machinery or large skilled workforce
•Significant handling stress and risk of post vaccination fungal
infections
•Local reactions
•Unsuitable for small fish
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.

Principle of vaccination: is the proactive induction of a protective

immune response by mimicking the natural interaction of an
infectious pathogen with the animal (vertebrate).

Vaccination is the administration of a vaccine to help the immune

system to develop protection from a disease.

Subunit vaccines are the purified antigens of the whole

organisms, that is used to trigger an immune response and
stimulate acquired immunity against the pathogen from which it
is derived .
Vaccination plays an important role in large scale commercial fish
farming and has been a key reason for the success of salmon
cultivation.

In addition to salmon and trout, commercial vaccines are

available for channel catfish, European sea bass and sea bream,
Japanese amberjack and yellow tail, Tilapia and Atlantic cod.

In general, empirically developed vaccines based on inactivated

bacterial pathogen have proven to be very efficient in fish.
Few viral vaccines are available but not for parasites.

However before such vaccines can be commercial and success,

several hurdles have to be overcome regarding production of
cheap but effective antigens and adjuvants while bearing in mind
environmental and associated regulatory concern.
Eg: Limited use of live vaccine
Lot of work on salmonids has been done on vaccine production
despite contribution to the total volume of farmed fish in the
world although crustaceans eg: shrimp, molluscons eg: clam and
oyster and aquatic plants are important in global aquaculture,
they did not possess an adopted and specific immune system.
Vaccines and disease control:
In all forms of intensive culture where single or multiple species are
recorded at high densities, infectious disease causing agents are
easily transmitted.
Fish such as carps often farmed in muddy ponds appear to be more
robust than for instance Atlantic salmon that are adopted to spend
their early life in clean freshwater.

Good environmental conditions are important to maintain a healthy

fish population for species reared in an open aquatic environment,
exposure to the pathogens is impossible to avoid.

Due to the effectiveness of pathogen transportation in water and

high density of animals used in commercial large scale farming
pathogen quickly spread within the population of cultured fish.
To reduce the use of antibiotics which are detrimental, vaccine are
alternatively used.

Fish immersion vaccine based on formalin inactivated broth culture

has proven to be effective against vibriosis in USA and similar vaccine
has been developed against salmon’s vibrio diseases but for
furuncolosis immersion vaccine proved to be infection against the
pathogen.

Injectable vaccines were developed in early 1990’s.

The excellent efficiency of oil adjuvant vaccine resulted in their

extensive use and an immediate reduction in the use of antibiotics
concurrent with 3 fold increase in production.
Recently the introduction of recombinant virus vaccines is done.

Good hygiene and linked stress reduction are key factors in

prophylaxis of infection disease and are also a necessity for the
optimal effect of vaccines.

Today vaccination is an integral part of most salmon farms and use of

antibiotics is very limited.

Until the early 1990’s the most fish vaccines were developed and
commercialized by small local companies
From 1990 till date five multinational animal health care companies
have acquired and specialized in the field of aquaculture vaccines
they are
 
Inter Vet International in Nether lands
Novartis Switzerland
Schering Plough Animal Health USA
Pharmaq Norway
Beyer Animal Health in Germany and Canada
 
Locally developed vaccines are also available in countries like Japan,
China, Russia, Spain and Germany.
Vaccine administration methods and factors affecting efficacy:
 
As oral vaccination by antigen included in feed gives poor and
inconsistent responses due to destruction in gut, but some methods
to protect the antigen by
entrapping in liposomes or alginate beads,
neutralizing gut fluids or application of biofilm vaccine are giving
promising results.

However the large quantity of antigen is required and protection

achieved is generally weak and of short duration.

Therefore the two main methods of vaccine delivery to fish are still
immersion in diluted suspension of vaccine or injecting into body
cavity by intraperitoneal injection.
An immersion vaccine is effective for a number of bacterial
pathogens and is cheap and easy to administer for small fish.

In contrast vaccination by injection is labor intensive and requires

fish to be over a certain size, vaccinating fry is difficult.

The advantage of injection vaccination is that volume of vaccine

needed is relatively low and every fish is vaccinated with the correct
dose.

For this professional vaccination teams and automated vaccination

machines are used.
Fish are cold blooded animals with body temperature that equals
their surroundings.
Depending on fish species and temperature, vaccination must be
performed with in certain period of time before their risk of
exposure to pathogens.
Onset of immune response is faster in warm water species than in
cold water species.
At an optimal temperature for Atlantic salmon 10- 12°C, antibodies
are typically not detected until at least 4 – 6 weeks after vaccination,
where as warm water species such as sea bass with an optimal
temperature of 22°C, can have detectable antibodies one week after
immunization.
In addition to temperature, stress caused by environment or
manmade factors such as photoperiod, seasonal changes, salinity,
heavy metals, crowdy, handling and transport affect responses.
Limitations in vaccine developments:
Although fish immune system is primitive compare to mammals,
there seems to be similarity then differences.
In the limited species of fish that has been studied, the major
antibody is immunoglobulin IgM like isotype that typically exist as a
tetramer in its secreted form.
Assays to measure specific T-cells responses are still in their infancy
in fish studies.
Lack of detailed knowledge on immune system in different fish
species limits possibilities to study both pathogen and vaccine
induced immunity.
As with all vaccines cost effectiveness in the field is essential
limitation to commercial vaccine development.
The difference in life stages, area of farming, species differences,
time of infection in growth of fish and high cost are another
limitation.
The apparent lack of maternal immunity in fish also limits the
possibility to protect off spring by parental vaccination.
Bacterial fish vaccines:
At present vaccines are available against many of the serious
bacterial diseases causing serious lose to fish farming industries.
The first commercially available vaccine for fish was Enteric Red
Mouth Disease (caused by Yersinia ruckeri) and Vibriosis in USA, in
late 1970’s.
These vaccines were based on inactivated whole cell formulations
and administer by immersion.
For each pathogen there are many serotypes, so one used for certain
species cannot be used for other.
A live attenuated Edwardsiella ictaluri vaccine in USA a few years ago
has proven efficacious by immersion of fish as early as 7-10 days post
hatching.
A live vaccine has also been licensed for using salmonids in North
America and Chile.
Viral fish vaccines:
Mostly available virus vaccines for aquaculture are based on
inactivated virus or recombinant subunit proteins.

Inactivated or killed viral vaccines are generally not efficacious

unless delivered by injection and as relatively high doses are
needed for achieving protection

Cost effective antiviral vaccines are difficult to achieve.

Live viral vaccines have been tested with good results in fish and
should be optimal regarding protection, administration and price.

However, Eco safety aspects of live virus, vaccines are considered

major drawbacks and currently hinder their use as commercial
vaccines.
Viral fish vaccines………………..
The first viral vaccines for fish was developed and produced against Carp
Rhabdovirus causing spring viremia of carps (SVCV) and based on two
inactivated strains SVCV emulsified in oil and administered by injection.

Although carp used for food and ornamental purpose in Asia widely, the
severe viral diseases such as Koi herpes virus, grass carp haemorrhagic
disease virus has been developed and used widely in china.

Another strain of Carp Intestinal Nephritis and Gill Necrosis vaccine are
available in Israel.
In salmon farming commercial vaccine for infectious pancreatic necrosis
virus (IPNV) is either inactivated cell culture propagated virus or
recombinant structural protein.
An inactivated viral vaccine against pancreas disease caused by aquatic
alpha virus is available in Ireland
Vaccine against infective salmon anemia caused by Orthomyxovirus is
available in Canada and USA.
Production of polyvalent vaccines:
For obvious practical reasons there reluctance to include more than
one vaccination during production cycle.
One of the most extensively used vaccines for salmons contains
antigen of 6 different pathogens and this number is likely to increase
with the emergence of new diseases.
However cost and demand for more complex products will sooner or
later will result in antigens being immunologically incompatible or
there may be limit to the amount of antigens that can be included in
water phase of oil water emulsion.
Furthermore the increasing number of antigen makes development,
production and registration of such vaccine more difficult.

Manufacturers may become reluctant to make small improvements as

any change requires huge effort in providing new documents for
licensing.
Production of polyvalent vaccines…….
It is also difficult to develop multivalent products that keep pace with
change in division situation in the field.
Because of the development it may be necessary to apply more than
one vaccination with fewer antigens.
Adverse effects following administration with adjuvant vaccine is
common problem for mammals and fish.
So new adjuvant with satisfactory efficacy and minimum side effects
are being developed in the industry.
For marine fishes other than oil adjuvants are required as it may be
necessary to prevent remnants of oil present in fish liver which is a
source for fish oil.
So delivery system of vaccine with modification in oral and immersion
therapy is the new field for development of vaccine therapy.
Vaccine development:
The development of vaccines typically follows a sequence of
activities that eventually leads to availability of a product.
Ideally a vaccine should be
Safe for fish as well as the consumer.
The vaccine should have broad strain or pathogen coverage
Provide 100% protection
Give a long lasting protection, preferably as long as the production
cycle
Be easy to apply
Be applicable in various species
Be cost effective
Be readily licensed or registered.
Of principle importance in the entire vaccine development process is
the precise identification of causative agent including the existence
and significance of serotypes and full understanding of the
epidemiology of the disease.
Identification of disease for selection of pathogen and
epidemiological information for establishing duration of protection
and to determine when fish should be vaccinated.
A combination of both will determine application method of choice
and vaccination schedule.
After development of challenge modal prototype, vaccine can be
developed and tested.
Different types of vaccine are possible
inactivated vaccine either adjuvanated or not,
live attenuated vaccine, subunit vaccine, recombinant vaccine,
synthetic vaccine or peptide vaccine or DNA vaccine.
Choice depends on degree of protection needed and duration and
final cost.