Fish and Aqua

Fish Vaccination

May 14, 2023

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Fish Vaccination

Aquaculture has been globally recognized as the fastest growing food production sector (FAO). The intensive farming of finfishes and shellfishes has led to an imbalance of optimal culture conditions, which shows increased susceptibility to infectious disease. Increased incidence of microbial diseases in aquaculture system is the major obstacle in the success of the industry. Use of antibiotics has attracted lot of criticism due to the issues like antibiotic residues, bacterial drug resistance and toxicity. In this present scenario, vaccination would be the best alternative to combat bacterial and viral disease for the sustainable aquaculture. The first report on fish vaccination was by David C. B. Duff and he is regarded as “Father of fish vaccination”.

Concept of vaccination

“Prevention is better than cure”

Vaccination is an easy, effective and preventive method of protecting fish from diseases. Vaccination is a process by which a protective immune response is induced in an animal by administration of vaccines. Vaccines are preparations of antigens derived from pathogenic organisms, rendered non-pathogenic by various means, which will stimulate immune system of the animal to increase the resistance to the disease on natural encountered with pathogens. Once stimulated by a vaccine, the antibody-producing cells, called B lymphocytes, remain sensitized and ready to respond to the agent should it ever gain entry to the body.

Importance of vaccination

� Vaccines are not the same as antibiotics and generally will not be effective for stopping a disease outbreak once it has begun.

� Vaccines are used to prevent a specific disease outbreak from occurring and are not a therapy.

� Its efficiency exists for a longer duration with one or more treatments.

� No toxic side effects and healthy fish have better growth performance.

� No accumulation of toxic residues

� Pathogen will not develop resistance.

� Theoretically it can control any bacterial and viral disease.

� No environmental impact.

Characteristics of vaccine

� The vaccine used should be safe and should not cause toxicity to the animal.

� The vaccine should have high immunogenicity.

� The vaccine should protect the animal for a longer period. In fish, duration of one year or life long protection can be said to be commercially effective for a vaccine preparation.

� The vaccine should be specific against a particular pathogen. It should not protect or favour the other pathogens.

VACCINES IN FISHES.

Vaccines is a biological preparation that improves immunity against the particular disease.
Vaccines contain a agent that identified a disease causing micro-organisms & is often made from weakened or killed forms of the microbes, its toxins or one of its surface proteins.
The agent stimulates the body’s immune system to recognize the agent as foreign, destroy it & remember it, so that the immune system can more easily recognize & destroy any of these micro-organisms that it later encounters.

History

First vaccine discovered by Edward Jenner for small pox.
Successful specific protective immunity in trout against Furunculosis in USA at 1942 by Duff.
Vaccine against bacterial disease in fish-1960.
Vaccine used to control Yersiniosis in trout in USA for sport fishing in 1970.
First commercial fish vaccines introduced in 1976 against ERM.
Vaccines against Vibriosis in late 1980.

Vaccines: “ Vaccines are preparation of antigen derived from pathogenic organism, 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”

Principal of vaccination

The major principal 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 safer immune response.

Characteristic of ideal vaccines

The vaccine used should be safe and should not cause toxicity to the animal.
The vaccine should have high immunogenicity.
The vaccine should protect the animal for a longer period. In fish, duration of one year or lifelong protection can be said to be commercially effective for a vaccine preparation.
The vaccine should be specific against a particular pathogen. It should not protect or favour the other pathogens.
It should be cheap.

Mode of preparation of fish vaccines

The bacterial fish vaccines may be categorized as follows.

i) Chemically or heat inactivated whole cells. These vaccines may be mono or polyvalent.
ii) Inactivated soluble cell extracts. i.e. Toxoids.

iii) Cell lysate

iv) Attenuated live vaccines, possiblygenetically engineered cells. There is a perceived risk that the vaccine strainmay revert to pathogenic mode.
v) Purified sub-cellular components, e.g. LPS. These vaccines require a detailed understanding of microbial biochemistry.

Methods of vaccine inactivation

There are several methods of inactivating bacterial cells for incorporation into fish vaccines.

i) Chloroform (3% v/v)
ii) Formalin (0.2-0.5% v/v)

iii) Phenol (0.3-3.0%)

iv) Heat (56° C or 100° C)
v) Sonication
vi) Lysis with NaOH at pH 9.5 or with SDS.

Commercially, the use of formalin has given encouraging results.

Types of vaccination

Killed whole cell vaccines/Inactivated vaccines: are the most common in aquaculture. 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. These have been of great use in controlling some of the important fish bacterial pathogens such as, anguillarum, V. salmonicida, V. ordalli, Y. ruckeri, and A. salmonicida. The vaccines obtained from bacteria called as bacterin. Bacterins are inactivated form of vaccines. Killed vaccines have been developed for some pathogenic fish virusessuch as infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosisvirus (IHNV), viral haemorrohagic septicaemia virus (VHSV) and spring viremia of carp virus (SVCV).
Live, attenuated vaccines: Live-attenuated vaccine is a suspension of attended live pathogens that are able to replicate inside the host and induce protective immune response but unable to cause disease.

They mimic the actual infection by pathogens and hence a small dose of vaccine is enough to induce long lasting protective immune response.
These live attenuated vaccines can induce both humoral and cell-mediated immune responses.
These are strong stimulants of cell-mediated immune response. These preferentially enhance T cell prolife rative response relative to B cell responses.
Some of the conventional live viral vaccines have been produced against VHSV, IHSV and IPNV.

Live vaccines also have the advantage that it stimulate the cellular immune response.
Edwardsiella ictaluri attenuated live vaccine has licensed in the USA.
Subunit vaccines are a more modern type of vaccine, developed from antigenic fragments that are able to evoke an immune response.
Subunit vaccines can be made by purification of parts of the actual micro-organism or they can be made in the laboratory using genetic engineering techniques.
An example is Intervet’s Compact® IPN used against infectious pancreatic necrosis virus infections of salmon in Chile.
Recombinant vector vaccines. A vaccine vector or carrier is a weakened bacterium into which harmless parts of genetic material from another disease-causing micro-organism have been inserted.
DNA vaccines are developed using plasmids containing information on the expression (of part) of the antigen of interest.
Synthetic/peptide vaccines are synthetically prepared antigenic epitopes in a suitable carrier or adjuvant.
Anti-idiotypic vaccines contain antibodies developed so as to mimic an antigen.

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.

Vaccination methods

Injection vaccination
Immersion vaccination
Oral vaccination

Injection vaccination

Initially, fish farmers may not favour injection vaccination as they fear that the stress resulting from the handling and injection of the fish will cause high mortality. Light anaesthesia of fish is needed for injection vaccination. This decreases the stress due to vaccination, prevents mechanical injuries and helps the fish to recover faster from the handling. When injection vaccination is performed properly, mortality immediately after vaccination should not exceed 0.25%. Injection vaccines can be administered by intramuscular or intraperitoneal (in the abdominal cavity) injection, but the latter is by far the most common. As intraperitoneal injection vaccination involves depositing the vaccine in the abdominal cavity, it is important that the needle should penetrate the targeted abdominal wall of fish by 1 to 2 mm. Short needles might deposit the vaccine in the musculature and cause inflammation and a bad immune response.

Injection vaccination has a number of major advantages that makes it a preferred vaccination method. Injection vaccination provides for a long duration of protection, i.e., for over a year, and it allows for multiple antigens to be combined in a single vaccine and, therefore, in a single administration. In addition, the fish farmer is assured that every fish in the population has received the vaccine and at the correct dose. The injection volumes per fish are usually 0.1 or 0.2 ml and give protection throughout the production cycle of most farmed species. Injections are in general superior to any other vaccine application method; however, from a practical point of view, they can only be applied to fish of 10g or more.

Advantages & disadvantages of Injection vaccination

Most common method of vaccine delivery in fish.
Highly efficient in generating both humoral (antibody) and cellular response.
Needs sophisticated machinery or large skilled workforce.
Significant handling stress and risk of post vaccination fungal infections.
Unsuitable for small fish.

Immersion vaccination

Skin epithelium and gills have mechanisms to protect fish in a broad as well as specific way. Immersion vaccination works on the ability of mucosal surfaces to recognize pathogens they had been in contact with. When fish are immersed in water containing the diluted vaccine, the suspended antigens from the vaccine may be absorbed by the skin and gills. Then, specialized cells, such as antibody-secreting cells, present in the skin and gill epithelium will be activated and will protect the fish when fish are exposed to the live pathogen at a later stage. In immersion vaccination, there are two application methods: dip and bath. In dip vaccination, fish are immersed for a very short duration, usually 30 seconds, in a highly concentrated vaccine solution, usually 1 part vaccine product to 9 parts water. With bath vaccination, fish are exposed for a longer period, usually one to several hours, in a lower concentration of vaccine.

Of the two alternatives, dip vaccination is more widely used since it facilitates fast vaccination of large numbers of fish (up to 100kg of fish per litre of vaccine). Immersion vaccination is widely used for vaccination of fry from 1 to 5 g. The limitations of immersion vaccination are that the duration of immunity is not very long and a booster vaccination is required when the disease prevails over longer periods. Also, the method is impractical for larger size fish due to cost-effectiveness and the stress that could be induced by vaccination.

Advantages & disadvantages of immersion 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.
Immersion tends only to stimulates the mucosal system with specific antibody secreting cells detected only in skin epithelium & gill after immersion vaccination.

Oral vaccination

With oral vaccination, the vaccine is either mixed with the feed, coated on top of the feed (top dressed) or bio-encapsulated. When vaccines are used as top dressing in feed, a coating agent is usually applied, either to prevent leaching of the antigen from the pellets or to prevent breakdown of the antigen in the acidic environment of the fish stomach. For sensitive antigens, various microencapsulation methods are being evaluated and tested. Bio-encapsulation is used where fish fry are to be vaccinated. In this case, live feed, such as Artemia nauplii, copepods or rotifers, are incubated in a vaccine suspension after which they are fed to the fry. Since these live organisms are non-selective filter feeders, they will accumulate the antigen in their digestive tract and as such, transform themselves into living microcapsules. Oral vaccination has the advantage that it is a very easy vaccine administration method with no stress to the fish. However, oral vaccines have a very short term stability once mixed with the feed. In most cases, only limited protection can be obtained and the duration of protection can be rather short.

Advantages & disadvantages of Oral vaccination

Vaccine mixed with feed
Easiest method for mass vaccination of all sizes of fish
Saves labour and avoids stress
Large quantities of antigen required
Requires all fish to be feeding
Protection generally weak and of short duration

Table: List of developed vaccines

VACCINES	SPECIES	DISEASE
Aeromonas salmonicida Bacterin	Atlantic salmon	Furunculosis
Vibrio anguillarum-Ordalii, Yersinia ruckeri Bacterin	Rainbow trout	Vibriosis, yersiniosis (enteric red- mouth disease)
Yersinia ruckeri Bacterin	Salmonids	Yersiniosis (enteric red-mouth disease)
Vibrio salmonicida Bacterin	Salmonids	Vibriosis
Vibrio anguillarum-salmonicida Bacterin	Salmonids	Vibriosis
Aeromonas salmonicida Bacterin	Salmonids	Furunculosis
Edwardsiella ictaluri Bacterin	Catfish	Enteric septicaemia
Spring viraemia of carp virus	Common carp	Spring viraemia of carp
Koi herpes virus (KHV) –Kh 3	Koi carp	Koi herpes virus (KHV) disease
Biofilm and free-cell vaccines of Aeromonas hydrophila	Indian major carps	Dropsy
Streptococcus agalactiae (group B) vaccine	Tilapia	Streptococcosis
Betanodavirus	Grouper	Betanodavirus disease

Infectious hematopoietic necrosis virus (IHNV)

DNA Vaccine

Recombinant G protein

Intramuscular injection

APEX-IHN

Types of vaccines

Killed vaccine

Vaccines containing killed micro-organisms, these are previously virulent micro-organisms which have been killed with chemicals or heat.

Inactivated vaccine

An Inactivated Vaccine is culture of an infection agent on a medium and then deactivated using heat or formaldehyde. Viruses are metabolically inert and they cannot be killed.

Attenuated vaccine

Some vaccines contain live, attenuated virus micro-organisms. These are live micro-organisms that have been cultivated under conditions that disable their virulent properties, or which use closely-related but less dangerous organisms to produce a broad immune response. They typically provoke more durable immunological responses and are the preferred type for healthy adults

Toxoid vaccine

Toxoids are inactivated toxic compounds in cases where these (rather than the micro-organism itself) cause illness.

Subunit vaccine

Rather than introducing an inactivated or attenuated micro-organism to an immune system (which would constitute a “whole-agent” vaccine), a fragment of it can create an immune response.

Conjugate vaccine

Certain bacteria have polysaccharide outer coats that are poorly immunogenic. By linking these outer coats to proteins (e.g. toxins), the immune system can be led to recognize the polysaccharide as if it was a protein antigen.

Experimental vaccines

A number of innovative vaccines are also in development and in use:

Recombinant Vector – by combining the physiology of one micro-organism and the DNA of the other, immunity can be created against diseases that have complex infection processes
DNA vaccination – in recent years a new type of vaccine called DNA vaccination, created from an infectious agent’s DNA, has been developed. It works by insertion (and expression, triggering immune system recognition) of viral or bacterial DNA into animal cells. Some cells of the immune system that recognize the proteins expressed will mount an attack against these proteins and cells expressing them. Because these cells live for a very long time, if the pathogen that normally expresses these proteins is encountered at a later time, they will be attacked instantly by the immune system. One advantage of DNA vaccines is that they are very easy to produce and store. DNA vaccination is still experimental.
T-cell receptor peptide vaccines are under development for several diseases .These peptides have been shown to modulate cytokine production and improve cell mediated immunity.
Targeting of identified bacterial proteins that are involved in complement inhibition would neutralize the key bacterial virulence mechanism.
While most vaccines are created using inactivated or attenuated compounds from micro-organisms, synthetic vaccines are composed mainly or wholly of synthetic peptides, carbohydrates or antigens.

Valence

Vaccines may be monovalent (also called univalent) or multivalent (also called polyvalent). A monovalent vaccine is designed to immunize against a single antigen or single microorganism. A multivalent or polyvalent vaccine is designed to immunize against two or more strains of the same microorganism, or against two or more microorganisms. In certain cases a monovalent vaccine may be preferable for rapidly developing a strong immune.

Adjuvants

Adjuvants are pharmacological or immunological agents capable of modifying the effect of other agents, such as drugs or vaccines. Adjuvants are virtually useless if given alone, but can serve to make a vaccine much more effective. When given together with a vaccine, the adjuvant will stimulate the immune system and increase its response to the vaccine. Exactly how adjuvants work remains unknown. Aluminium salt, virosomes and certain oils are all commonly used as adjuvants for vaccines.

Vaccine delivery system

There are three methods of vaccination in fish.

Injection delivery system.
Immersion delivery system.
Oral delivery system.

Injection delivery system

Injection delivery system is an effective way of inducing antibody response in fish. The injection may be intraperitoneal or intramuscular. But the major setbacks are, it is time consuming, labour intensive and consequently expensive to administer to large number of animals. During the course of injection, handling stress affects their ability to produce optimal immune response. For safe handling and injection, the fish should be of reasonable size. Fry cannot be vaccinated by means of injection. Because of these limitations injection delivery system of vaccines are used in fishes of high unit value to absorb the cost of procedure.

Immersion delivery system

Immersion delivery system of vaccines is a potential commercial process. It is a hyperosmotic immersion technique, where prior to immersion in antigen solution fish are dipped for a short time in a hyperosmotic salt solution which enhances the uptake of antigen. The advantages are, large number of fish can be vaccinated at the same time and it can be used to vaccinate fry of any size above the critical size of immune responsiveness. The main disadvantage of this method is the fish is subjected to stress. Spray vaccination is a variant of direct immersion where antigen in sprayed under pressure on to the fish, as they are propelled along a shallow channel.

Oral delivery system

Oral delivery system through feed is a potentially useful method of vaccination. Besides its simplicity, it is a method of choice for eliciting immune response to enteric pathogens. This method do not cause stress to the fish. It can be used to vaccinate the fish of any size and requires no extra time or labour than normal farm husbandry. The main limitations of this method are, to obtain effective immunity it is necessary to give a large dose of antigen (i.e.) the total amount of antigen needed is much more higher than injection and immersion delivery systems and the immunity which the oral delivery system provokes is poor and not as long lasting as injection delivery system. The poor response is due to poor antigen delivery to immune responsive sites. Due to low pH and high enzymatic activity the vaccine is destroyed in the foregut and causes poor antigen delivery to hind gut and other lymphoid organs of fish. To overcome this problem encapsulated vaccines can be used. Recently biofilm of bacterial pathogen has been evaluated successfully for oral vaccination of fish with high antibody tire and protection. The glycocalyx of biofilm helps to resist the vaccine destruction in the foregut favouring better antigen delivery to immune responsive sites in the hind gut.

How to achieve optimal effects of vaccines

Using efficient vaccines and administering them correctly is not the only factors affecting the effects of vaccines. Optimal efficiency of vaccines can be achieved by proper fish management practices. Optimal conditions and adequate nutrition are very important and one must also strive to expose the fish to as little stress as possible. The efficiency of a vaccine largely depends on the condition of the immune system and exposing fish to factors that might harm their immune system are therefore highly unadvisable.

List of fish vaccines developed

VACCINES	SPECIES	DISEASE
*Aeromonas salmonicida* Bacterin	Atlantic salmon	Furunculosis
**Vibrio anguillarum-Ordalii-Yersinia ruckeri Bacterin**	Rainbow trout	Vibriosis, yersiniosis (enteric red- mouth disease)
*Yersinia ruckeri* Bacterin	Salmonids	Yersiniosis (enteric red-mouth disease)
*Vibrio salmonicida* Bacterin	Salmonids	Vibriosis
*Vibrio anguillarum-salmonicida* Bacterin	Salmonids	Vibriosis
*Aeromonas salmonicida* Bacterin	Salmonids	Furunculosis
*Edwardsiella ictaluri* Bacterin	Catfish	Enteric septicaemia
Spring viraemia of carp virus	Common carp	Spring viraemia of carp
Koi herpes virus (KHV)	Koi carp	Koi herpes virus (KHV) disease
Biofilm and free-cell vaccines of Aeromonas hydrophila	Indian major carps	Dropsy
*Streptococcus agalactiae* (group B) vaccine	Tilapia	Streptococcosis
Betanodavirus	Grouper	Betanodavirus disease

Koi herpes virus (KHV)	Attenuated viral vaccine	Attenuated virus	Immersion or injection	KV-3 (also known as Cavoy)
Infectious pancreatic necrosis virus (IPNV)	Subunit vaccine	VP2 and VP3 capsid proteins	Oral	AquaVac® IPN Oral

Recent development in fish vaccinology

During the last two decades vaccination has become established as an important method for prevention of infectious diseases in farmed fish, mainly salmonid species. So far, most commercial vaccines have been inactivated vaccines administered by injection or immersion. Bacterial infections caused by Gram-negative bacteria such as Vibrio sp., Aeromonas sp., and Yersinia sp. have been effectively controlled by vaccination. With furunculosis, the success is attributed to the use of injectable vaccines containing adjuvants. Vaccines against virus infections, including infectious pancreatic necrosis, have also been used in commercial fish farming. Vaccines against several other bacterial and viral infections have been studied and found to be technically feasible. The overall positive effect of vaccination in farmed fish is reduced mortality. However, for the future of the fish farming industry it is also important that vaccination contributes to a sustainable biological production with negligible consumption of antibiotics.

Indian Immunologicals first to venture into fish vaccines

Hyderabad-based vaccine manufacturer Indian Immunologicals Limited (IIL) has announced a partnership with Central Institute of Fisheries Education (CIFE), Mumbai, an Indian Council of Agricultural Research (ICAR) Institute for the commercial development of vaccine against common bacterial diseases in freshwater fishes.

IIL has forayed into Aqua business in October 2022 by launching products for aquaculture health market dealing with pond management and fish or shrimp gut management.

Speaking on the occasion, Dr K Anand Kumar, Managing Director, Indian Immunologicals Limited said, “IIL has introduced several innovative veterinary vaccines first to the world, like Porcine Cysticercosis vaccine, FMD+HS+BQ combination vaccine and Theileria vaccine. IIL is again the first in India to get to fish vaccines.”

CIFE will provide technology for two inactivated bacterial vaccines, one for Columnaris Disease, a serious condition affecting numerous freshwater fish species, and other for Edwardsiellosis that cause high degree of mortality, leading to severe economic losses. Both the diseases are extremely common in freshwater fishes and is generally considered to be ubiquitous.

Dr Priyabrata Pattnaik, Deputy Managing Director, Indian Imunologicals Limited added “IIL is planning to introduce vaccines and immunostimulants with tech transfer from various fisheries institutes under ICAR. Several fish vaccine candidates are currently being evaluated by IIL for commercialisation.”

Dr CN Ravishankar, Director and Vice Chancellor of ICAR-CIFE said “In support to India’s Blue Revolution, I am glad that CIFE and IIL have come together to partner in developing India’s fist bacterial fish vaccine”.

Currently there is no fish vaccine available in India on a commercial scale to prevent aquaculture infections.

In addition to optimizing husbandry and general management practices, use of vaccines is still limited, is becoming more widespread in certain sectors of aquaculture for disease prevention. A number of vaccines have been in use by the salmonid industry for decades. However, commercial vaccine development for other aquaculture sectors, including producers of warm water fish, is still quite limited. Greater demand by producers and increased levels of research and interest by manufacturers is helping to make vaccination a more viable option. Currently, vaccines are available for some economically important bacterial and viral diseases. Vaccines for protection against parasitic and fungal diseases have not yet been developed. Vaccination should be considered part of a comprehensive fish health management scheme, and not the only solution for a disease problem.