INTRODUCTION
A vaccine is any biologically based preparation intended to establish or to improve immunity to a particular disease or group of diseases. Vaccines have been used for many years in humans, terrestrial livestock, and companion animals against a variety of diseases. One analogy used for vaccines is that of an insurance policy (Komar et al, 2004). A vaccine, if effective, can help prevent a future disaster from being a major economic drain. But vaccines, like insurance, have a premium, or cost. The producer must weigh the cost in materials and labor against the risk and cost of a disease outbreak to determine whether vaccination is warranted.
When actual vaccine effectiveness is also unknown, this makes decision-making even more difficult. Consultation with a fish veterinarian or other fish health specialist will be helpful when examining cost vs. benefit of a particular vaccine Ingunn et al (2005) reviewed vaccines for fish in aquaculture and the he concluded that development of commercial vaccines for specific fish pathogens is helpful for successful fish production from aquaculture. Fish over a certain size/ age threshold are capable of mounting both antibodies mediated and cellular immune response. The specific or adaptive immune response has two types (vide fig. 1).
Exposure to an antigen or a vaccine results in stimulation of lymphocytes responsible for antibody production or CMI. Vaccines work by inducing a protective immune response which by the virtue of memory cells persists for a long time. Commercial vaccines to combat infectious by Yersinia ruckeri and Vibrio anguillaraum are available and experimental vaccine against Aeromonas salmonicida, A. hydrophila, Pseudomonas anguilliseptica has been found to be effective against laboratory challenges. The objective of vaccination is to prevent disease. The main purpose of vaccination is to increase the specific immunity to infections to which the vaccinated fish are likely to be exposed.
History
The first vaccine for aquaculture, a vaccine for prevention of yersiniosis in salmonid fish, was licensed in USA in 1976. Since then the use of vaccines has become well established and cost effective method of controlling various infectious diseases and expanded to new countries and new species simultaneous with the growth of the aquaculture industry. The first report on fish vaccination was given by David C. B. Duff and he is regarded as “Father of fish vaccination”. Optimal husbandry and general management-including biosecurity, nutrition, genetics, system management and water quality—are critical for maximizing aquatic animal health. However, all facilities are vulnerable to disease outbreaks because many pathogenic (disease-causing) organisms (e.g., bacteria, viruses, fungi, and parasites) are opportunistic and present in the environment, or may be found on some fish that are not showing signs of disease (carriers). When disease outbreaks occur, diagnostics are conducted to determine the cause and then the fish are given; an oral treatment, an immersion (a dip or a bath), or, in rare cases, an injection treatment. Costs incurred from delayed production and growth, treatment chemicals, mortalities, and labor can be significant. In many cases, when fish are no longer eating, treatment options become much more limited and treatment may no longer be effective. It makes sense, then, that prevention of disease is preferable to disease treatment (Grisez and Tan, 2005). Two approaches to disease prevention that historically have been used in other animal industries are vaccines and immunostimulants. Both approaches have been used successfully in some aquaculture industries, and should be considered fish health management options.
Concept of vaccination
The word “vaccination” originated from the work of Jenner who inoculated a boy with infectious material from cowpox in order to induce immunity to smallpox (Jenner, 1798). He called the process “vaccine inoculation”, later “vaccination”. Later Louis Pasteur suggested that the word “vaccination” should be a general word for preventive inoculation of microorganisms as a tribute to the work of Jenner (Fenner et al, 1997).
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. In vertebrates survivor of a pathogen infection become resistant to subsequent infection by the same pathogen.
This resistance is called adaptive immunity and it is specific to the challenging pathogen and persists for a relatively longer period of time. It is based on adaptive change in body lymphocyte population resulting from exposure to foreign molecules constituting the pathogen. Specific and memory of the adaptive immune system are the two key elements of vaccination. Several researchers Evelyn (1997), Muiswinkel (2008), Snieszko (1970), Newman (1993), Toranzo et al (2009) and Gudding et al, reviewed fish vaccines as topic of their review articles. Douglas and Anderson in year 2004 studied the use of Yersinia ruckeri specific bacterial strain for vaccination.
Characteristics of fish vaccine and importance of vaccination
The important characteristics of fish vaccines includes 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 and vaccines are easily licensed.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 as well as has no adverse environmental impact.
Development of fish vaccines
Today, vaccination is an integral part of most salmon farms and the use of antibiotics is very limited due to emergence of antibiotic resistant organisms. The development of fish vaccines is, as with the development of human and veterinary vaccines, an ongoing interaction between academia, the pharmaceutical industry and regulatory authorities. Until the early 1990s, most fish vaccines were developed and commercialized by small local companies. During the 1990s and to date, five multinational animal health companies have acquired, or formed, joint venture companies with the smaller companies specializing in the field of aquaculture vaccines.
The major producers of fish vaccines are Intervet International (The Netherlands), Novartis Animal Health (Switzerland), Schering Plough Animal Health (USA), Pharmaq (Norway: was part of Alpharma Animal Health until 2004) and Bayer Animal Health (Bayotek)/Microtek, Inc. (Germany/Canada). The major commercial markets for these companies are currently the salmon and trout industries in Northern Europe, Chile, Canada and the USA where the value of a healthy population justifies the price paid per vaccine dose. The fish vaccine is developed by following steps—
1. Recognition and epidemiology of the disease
In-depth knowledge of the epidemiological factors of a particular disease, including its occurrence and spread, target species, biology of the host and pathogen, pathogenicity and pathogenesis, and prevalence and incidence in a population is important to understanding the disease and to identify the targets for a protective immune response. The current host and geographic range of the disease, as well as its potential for further dissemination, needs to be evaluated.
2. Isolation of pathogen and characterization of disease
Isolation of presumptive pathogens and a full description of the disease symptoms are critical. It is not unusual for a particular disease of fish to have more than one etiological agent or to be exacerbated by another disease. Furthermore, it is important to work with virulent strains recently isolated from moribund host species showing typical clinical signs of the disease.
3. Identification of species, strains and serotypes of the causative agent
Absolute identification of the causative pathogen using the most up-to-date techniques (including LPS stained gels, gel electrophoresis and slide agglutination tests) is necessary. Species of bacterial and viral pathogens often show variation in serotype groupings. The possible geographic and host specific differences of a given pathogen must be considered.
4. Key virulence factors and protective antigens
Identification of key virulence factors and protective antigens, followed by the development of an appropriate challenge model, which should simulate the natural disease condition as much as possible and must consistently provide reproducible and statistically significant data, must be developed.
5. Experimental production
Lab scale production of protective antigens and formulation of an experimental vaccine should be developed. Due to interaction among antigens, it is important to determine the optimal proportions of multiple antigens to ensure the best and most complete protective immune response.
6. Production/downstream processing/quality control/GMP
Both production and downstream processing can be critical issues. Certain technical processes are difficult, costly or even impossible to scale up from the laboratory bench to the production volumes needed. The practicality and convenience of packaging and services are further value added product characteristics.
Types of fish vaccines
1. Killed Whole Cell Vaccines
It is a suspension of heat or chemical – killed pathogens that are able to induce specific protective immune response against pathogens when administered into host. Some vaccines are formed for bacterial pathogens such as V.angullarium, V.salmonicida and V.ordalli. All the killed vaccines are formalin inactivated whole cell vaccines administered with or without adjuvant.
These bacterial vaccines are highly immune-protective and are cheap to produce. Killed vaccines have been developed for some pathogenic fish viruses such as infectious pancreatic necrosis virus, infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus and spring viremia of carp virus.
2. Live – Attenuated Vaccines
A live attenuated vaccine is a suspension of attenuated live pathogens that are capable to replicate inside the host and induce protective immune response but are unable to cause disease. They mimic the actual infection by pathogens and hence a small dose of vaccine can induce long lasting protective immune response. These vaccines can induce both humoral and cell mediated immune response.
These preferentially enhance T cell proliferative response relative to B cell response. Some of the vaccines are produced against VHSV, IHNV and IPVN. Ronen (2004) stated that attenuated viral vaccines is against KHVD and based on attenuated Cyprinid herpesvirus3 ( CyHV-3) Koi herpes virus (KHV) and gill necrosis virus.
3. Recombinant DNA Based Vaccines
It is defined as formation of new combinations of genetic material by insertion of nucleic acid molecules produced outside the cell via a vector system. These types of vaccines are termed as recombinant DNA based vaccines or new generation vaccines.
Four different types of vaccines are there
a. Recombinant immunogenic protein vaccines or epitopes purified from vectors carrying the gene of interest produced in prokaryotes or eukaryotes expression system.
b. Peptide vaccines
c. Live vaccine produced by defined genetic manipulation and microbial vectors carrying gene coding for immunization protein
d. DNA vaccines.
A . Recombinant Protein Vaccine
Production of a recombinant protein vaccines starts with identification of the immunogenic subunit or protein from the pathogens of interest and verification of its immunogenicity in vitro and in vivo. The vector system usually used to express recombinant proteins are viruses or bacterial plasmids. Both prokaryotes and eukaryotes expression system have been used to produce fish viral, bacterial and parasitic antigens, but most widely used being the prokaryotic system.
Recombinant protein vaccines produced in prokaryotic system
v Infectious pancreatic necrosis virus
v Infectious haematopoietic necrosis virus
v Viral haemorrhagic septicemia virus
Fish viral vaccines produced in eukaryotic cell
So many eukaryotic cells such as insect’s cells have been used as the recombinant protein expression host with baculovirus as vector to produce fish viral protein. IPNV polyprotein and IHNV glycoprotein have been expressed in baculovius and are found to be antigenic but their efficacy in fish immune protection has not been tested. Dhar et al, (2014) studied the viral vaccines for farmed fin fishes as the topic of their review article.
Recombinant protein vaccine for fish bacterial disease
Some of the fish bacterial extracellular products such as proteases and the iron – regulated outer membrane proteins have been the target antigens for recombinant vaccines development. Fusion protein of 70 kD serine proteases of Aeromonas salmonicida has been cloned expressed and used as a prototype vaccine. Recently it has been reported that native major adhesion of Aeromonas hydrophilla which is a 43 kD outer membrane protein has the potential of been used as fish vaccines.
Recombinant protein vaccines for fish parasitic diseases
Many of the fish parasite pathogens such as Ichthyophthirius multifiliis, Amyloodinium acellatum, Cryptobia salmositica, Lepeophtheirus salmonis are potential candidates for vaccine development
B. Peptide Vaccine
Peptide vaccines comprises of synthetic peptides that are able to induce protective immune response when administrated into the host. For the production of peptide vaccines it is necessary to identity immunogenic regions or ‘epitopes’ on the antigens protein. The term epitope refer to a stretch of 6 -8 amino acids on antigens that specifically binds to antibodies or to receptors on immune T cell. The epitopes that binds to the antibody produced by specific B cells are called as B- cell epitopes while those recognized by receptors on the surface of activated Tcells are termed as T-cell epitotes. Monoclonal antibodies are indispensible to identify the B-cell epitopes.
C. Genetically Modified Live Vaccines
Pathogens with the defined genetic manipulations or microbial vectors carrying the gene coding for immunogenic protein can be used as live vaccines. Live vaccines replicate inside the recipient host resembling the natural infection and thus induce strong immunity. This kind of vaccine is reported to be highly immunogenic than the non replicating vaccine products. Live vaccines have been used against some of the fish bacterial pathogens such as Aeromonas salmonicida and Aeromonas hydrophila. So many techniques such as homologous recombination, chemical mutagenesis and transposon mutagenesis are used to produce mutant bacteria those are avirulant and capable of being used as live vaccines.
D. DNA Vaccines
A DNA vaccine against IHNV was the first effective DNA vaccine tested in fish after that DNA vaccines were tested against various fish viruses like IPNV ( Mikalsen et al, 2004), VNNV (Skall et al, 2005), SVCV ( Vembar et al, 2010). DNA vaccines consist of a suspension of bacterial plasmids carrying the gene coding for the immunogenic protein under the control of eukaryotic promoter. Heras et al, (2010) in their study orally delivered the IPNV DNA vaccines expressing VP2 antigen and the findings showed 80% Relative Percent Survival upon challenge by an infectious homologous virus in rainbow trout fry of 1-2g size. Aoki et al, in year 2011 evaluated efficacy of 2 DNA vaccines derived from pathogenic viruses such as HRV and VHSV through gene expression profiles and they also studied the gene expression profile of vaccinated and unvaccinated fish evidences that DNA vaccines plays very important role as immunostimulator or immunity enhancer in the host.
The basic attributes of a DNA vaccine include
1. An origin of replication suitable for producing high yield of plasmid in E. coli,
2. An antibiotic resistant gene to confer antibiotic selected growth in E. coli,
3. A strong enhancer/promoter and 4. A mRNA transcript termination/polyadenylation sequence for directing expression in mammalian cells.
The constructed plasmids are grown in E. coli, purified and suspended in saline and introduced into the host either by intramuscular injection or using a gene gun.
Factors affecting response to vaccine
- The age and size of fish: - Some salmonids species cannot develop any specific immune response until they reach 1.5 g live weight, weight apparently more important then age.
- Pre-existing diseases: - The effects of pre existing disease have no response on to a vaccine which depends on the type of disease. Some disease may affect either the uptake of vaccine or the immunological response.
Fish suffering from some disease will not feed and so will not spontaneously ingest any orally administrated vaccine. For immersion vaccines gills are the route uptake, so any gill pathology, whether due to water pollution, microbial infection or metazoan infestation will reduce uptake.
- For immersion vaccines the concentration, exposure time and use of any hyper osmotic infiltration procedure affect the response.
- For oral vaccines the method of incorporation in feed
- The water temperature – which should be monitored throughout the interval between vaccination and sampling.
- For euryhaline species the salinity
- The nutritional status of fish
- Any stressors occurring during the interval between vaccination and sampling – which will depress immune response
Administration of vaccine into fish is a major determinant in the success of immunization. Although the vaccine is a highly immunogenic so lack of proper route of administration may result in failure of immunization. Adjutants, immune-stimulants can be used in combination with vaccine to enhance the efficacy of the vaccine.
Route of administration
1. Oral Vaccination
Oral vaccination or presentation to fish of antigens adsorbed on the food is potentially the most useful method of vaccination available. Beside its simplicity it is a method of choice for eliciting immune response to enteric pathogens. It is non stressing, can be used to vaccinate the fish of any size and require no extra labour or time than normal farm husbandry. The efficacy of oral vaccination in proportion to the antigenic mass used is low and it varies according to the nature of the antigen and the vaccine formulation. The immunity which oral vaccination provokes is usually poor and not as long lasting as that provoke by injection.
2. Immersion Vaccination
Immersion of fish in antigen solutions emerged as a potential commercial process. It is a hyper osmotic immersion technique where prior to immersion in antigen solution fish are dipped for a short time in a hyper osmotic salt solution, which enhances the uptake of antigens. it can easily be adopted to vaccinate large number of fish and it can be used to vaccinate fry of any size above the critical size for immune responsiveness. It is stressful to fish. Factors affecting uptake are time, anatomy, fish size, particle size and adjuvant.
3. Spray Vaccination
It is seen as a variant of direct immersion where antigen is sprayed under pressure on to the fish, as they are propelled along a shallow channel.
4. Injection Vaccination
Injection of antigens (intraperitoneal or intramuscular) is an effective way of provoking an antibody response in fish and stimulates a better immune response than oral and immersion administration and it is possible to produce efficacious multivalent vaccines. Injection dose is normally independent of size of fish whereas oral and immersion vaccines depend on it. It is a time consuming, labour intensive and consequently expensive to administer vaccine to large number of fish.
Optimal effects of vaccines
Proper fish health management with good hygiene and limited stress are key factors in the prophylaxis of infectious diseases and are also a necessity for the optimal effect 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.
Main fin fish diseases controlled with vaccination
The major causative agents of infectious diseases in fin fish aquaculture include fungi 3.1%, Parasites 19.4%, Viruses 22.6% and Bacteria 54.9% (McLoughlin et al, 2007). The main groups of farmed fish which are routinely vaccinated are Atlantic salmon (Salmo salar), Rainbow trout (Onchorhychus mykiss), and now, increasingly, Atlantic cod (Gadus morhua). Ahmed and Ashram (2002) in his research stated that immunization of tilapia with formalin- killed whole culture vaccine through intraperitonial route gives active immunization of tilapia against Aeromonas hydrophylla. Christopher et al, (2014) reviewed the experimental trials on the effectiveness of inactivated, attenuated and sub-unit vaccines against Streptococcus iniae, S. agalactiae, Vibrio spp., Aeromonas hydrophylla, Edwardsilla tarda and Francisella asiatica in tilapia. They also tested the protective efficiency of bacterial vaccines against Vibrio anguillarum in Asian sea bass
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. 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 are to be used for booster vaccination. Development of new non-mineral oil adjuvants lacking side effects is an important need of today.