T- Helper Types and Their Associated Cytokines
6.4 ADVANTAGES AND LIMITATIONS IN AQUACULTURE
90 Fish Vaccines immunization; hence many of today’s vaccines are delivered this way. This can be done manually with a needle or by using technologies like compressed air. The length of protection is longer in this method than in the immersion method. The vaccine is not diluted in this method. It is also supplemented with other substances, including adjuvants, carriers, bacterial cells, bacterial anti- gens, and others that aren’t available through standard immunization methods (Dadar et al., 2017).
Antigens for injectable vaccines can be conveniently kept at 4°C. Water-in-oil (w/o)-based injectable vaccinations are often delivered via intraperitoneal injection, whereas DNA plasmids are typically delivered via intramuscular injection (Embregts and Forlenza, 2016). Both humoral antibody and cellular antibody and cellular cytotoxic responses are highly efficient with injection.
An additional benefit of the injection vaccination methodology is that a multivalent vaccine can deliver numerous antigens from various infections simultaneously. But this method is not practiced commonly or recommended for fishes that are small in size or less than 5 g in weight as there are numerous risks in this way of administering the vaccine. As fishes would be very small, it would take much time to vaccinate enough biomass of fishes. This will be a highly complex process. Small fishes may also suspend their feeding activity temporarily. Adhesion formation and penetration of the intestines may also happen. Injury at the injecting point is also highly possible in very small- sized fishes, which can quickly worsen into another kind of infection. Moreover, w/o-based inject- ing vaccines have been caused by local side effects, which include many problems such as tissue inflammation. More issues such as necrosis and adhesion have been common occurrences in this process, decided by the type of adjuvant applied. Furthermore, many deaths have been reported because of ill-handling and stress on the body during the injection. One restriction associated with injectable vaccinations is that they can’t be used several times during the fish production cycle.
They cannot be used in early life stages because the immune system is still immature. During those stages, their body is delicate, and they can’t take the handling stress during the injection of the vac- cine. Frequently necessitates the use of sophisticated machinery or a big, experienced team.
Intraperitoneal injection can be administered both by manual and automatic methods. In the process of manual injection, the fish are anesthetized before injection. The injection is usually administered by an air-powered syringe. The vaccine can be administered to 1000–1200 fish in the manual method, and about 7000–9000 fish can be vaccinated using the automatic method. In the automatic method, fishes have to be of bigger and similar size for vaccine administration (Plant and Lapatra, 2011).
Advancements in Vaccine Delivery and Methods of Vaccination 91
and more common. As the density of culture species are very high, the loss incurred due to any infectious disease would be a large fraction for the aquaculture farmer. Such disease can also enter farms if the conditions and the species are in a healthy state and eliminate a large number of culture species. Intensifying culture means there is a constant looming break out of infectious disease by emerging and re-emerging pathogens even if all parameters have been maintained, so the occur- rence of diseases cannot be predicted (Vinitnantharat et al., 1999). Mixed infections are also occur- ring (Hwang et al., 2020). US$ 50 billion are lost every year in aquaculture due to inadequate health management of fishes (Rather et al., 2011). Such diseases directly affect the livelihood of people and also the health of people consuming fish. For sustainable aquaculture practices to continue, they have to stay profitable for the farmer. Huge losses would be a significant deterrent for anyone who has invested in the venture.
Antibiotics provide useful means to fight against many bacterial diseases, but continued use of antibiotics has induced the development of bacteria resistant to them, thus making them ineffective.
Accumulation of antibiotics also causes pollution (Karunasagar et al., 1994). The treatment thus becomes more costly and complex. Chemotherapy in fishes is not possible as diseased fish does not take feed properly (Hwang et al., 2020). Such a scenario has forced people to change strategy as to prevent the diseases altogether rather than treatment.
After many kinds of measures were tried, the fish diseases continue to surprise scientists and the farmer, and they continue to be a major reason for huge aquaculture losses throughout the globe. Drugs such as antibiotics have been applied as a solution, but they have been successful only to a threshold in many instances. Though the use of antibiotics has led to more understand- ing of fish health, improper usage has also led to undesirable results such as food safety hazards, drug resistance and safety issues are common deterrents in their case (Sneeringer et al., 2019). By using different prophylactic measures, managing fatal infections can be achieved. Vaccination had a major impact on controlling outbreaks in culture fishes. To date, most vaccines for fish have been developed against bacteria, and lesser numbers for viral infections (Embregts and Forlenza, 2016).
Improvement of vaccines for fish is an ongoing process. There is an unknown number of diseases for which vaccines have to be developed. Fishes are poikilotherms, so vaccine development includes factors such as species, delivery measures, routes, etc. (Dadar et al., 2017).
Vaccination in fish is being practiced for over 50 years and provides stability and sustainabil- ity to aquaculture. A fish vaccine means something which has antigen or induces their produc- tion in the organism. An ideal fish vaccine would induce strong immunity for long periods, is not very costly to manufacture, has little or no side effects, and its method is not too complex (Ma et al., 2019). Vaccination is based on the principle of isolating, inactivating, and injecting, as stated by Louis Pasteur. Vaccines aim to provide safer food as well as maintain the environment of the surroundings. The advantages of immunization are manifold. With new techniques developing in microbiology and biotechnology, there is the rapid development of vaccines far from conventional ones. Sudden use of antibiotics in Norwegian Salmon after starting immunization has happened (Ma et al., 2019).
The first vaccination in fish was done in 1938 by Snieszko and co-workers in the carps against Aeromonas punctata by using killed bacteria, which induced immunity. But the process was too complicated to be replicated on the average aquaculture farms and people would discard the prac- tice (Gudding and Van Muiswinkel, 2013). The first fish vaccine in the US was licensed in the 1970s and commercialized in the 1980s (Shao, 2001). Now, vaccines for 17 fish species are developed against 22 bacterial and six viral diseases. Large-scale vaccination was mostly done in Salmons in earlier times, but now other species also are taken. Vaccination is being done in more than 40 countries and is being given in several ways which include immersion, injection, and oral, among others (Brudeseth et al., 2013).
Immersion vaccination has been proven a convenient method. It also includes the dip method, bath method, and spray immersion. Fishes are simply immersed in a dilute vaccine for a short time and sent back to the culture area. Exposure duration can be between 30 and 60 seconds. But this
92 Fish Vaccines has some limitations too, as, in cases of those fish that cannot be taken out of the culture area, this method can work when they are going to be put there. After that, they are not taken out. But, in large-sized fish, it helps in reducing the cost of the process. Smaller size fishes also can be immu- nized in large numbers at the same time. As it has a diluted version of the vaccine, some part of it would be lost, but it doesn’t mean the efficacy is also reduced.
Intraperitoneal injection is the most effective method of vaccination. It allows the usage of adju- vants which will increase the time frame of its efficacy. Oil adjuvant is very commonly used in this case. Adjuvants are highly efficient and useful substances being put to use in vaccination. They form a part of the delivery mechanism of vaccines and make them many times more potent than earlier.
They target specific cells or their organelles. They are abundantly taken up by mucosal surfaces and can easily transport through different barriers. Fishes are anesthetized and injected, then returned.
Commercial operations use repeating injection guns that can inject 1000–2000 fish in just an hour.
But this method is time taking and also requires skilled personnel, which is rare and also would increase the production costs by a number. Injecting smaller fish is not practical, as they may be too stressed due to handling. Wounds due to injection might not heal fully, leading to the loss of the fish (Vinitnantharat et al., 1999).
Oral vaccination means the fish can get the vaccine through their feed. This means extra costs of any kind can be avoided. No skilled labor is needed, no extra time would be spent on the same activ- ity, and fishes of all sizes will be immunized. The vaccine is incorporated in the feed, so it needs some kind of protection also as it would be put in water thus leaving only some part of the vaccine available to the fish. Among the three most common methods, this method is the least potent due to this reason. The digestive system of the fish is a harsh and dynamic path through which this vaccine has to pass. So, preparing feed having adequate protection or covers make the method less smooth. In the intestines, the microfold cells are integral to the absorption of such vaccines to Peyer’s patches and further into lymphoid follicles found in the mucosal layer. Microparticles are taken by the microfold cells spontaneously (Embregts and Forlenza, 2016).
The aim of vaccination is to induce immunity by activating the immune system. There are two components of immunity, innate and adaptive. Innate immunity is more reactive and acts sponta- neously on sensing any infection. Surfaces such as skin, alimentary tract, gills, and mucous mem- branes form this arm of fish immunity. Growth inhibitors, for example, interferons, phagocytes, macrophages, and lysins, among numerous more constitute innate immunity, which is very rapid in its action. The adaptive immunity takes more time to get activated, but it gives specific memory, which is needed for the complete removal of the pathogenic substance. The adaptive immunity is made up of the humoral immunity, CMI, and immunological memory of the body. Humoral immu- nity, like other mammals, is due to the production of immunoglobulins, which have been found in three types, IgM, IgD, and IgT (Hu et al., 2010). CMI is due to T-cell involvement to combat pathogens whenever detected. The immunological memory is, obviously, the basis for identifying the antigen and making the required changes in the lymphoid cells (Secombes and Ellis, 2012).
Immunization should result in activating both kinds, otherwise, it cannot be called successful by not having a lasting effect. For further enhancing the impact, adjuvants and nanoparticles are being used along with the traditional or conventional vaccines, which enhances the delivery system by preventing the degradation and loss of the integral structure and properties of the antigen. It has been seen and proven that without their application, the effect of vaccines is not satisfactory. Some studies have also shown that nanoparticles themselves act as an antigen, which is not desired and complicates the matter (Zaman et al., 2013).
6.4.1 adVantages
Vaccines for most diseases have not been developed, and many are still in progress. The commence- ment of practicing immunization has not completely eradicated infections, but it has significantly reduced mass mortality. Nanoparticles are now part of most vaccine formulations. They act by two
Advancements in Vaccine Delivery and Methods of Vaccination 93
mechanisms, either as an adjuvant or as becoming a part of the delivery system. Nanoparticles that act as the delivery system will deliver the antigen to the cell targeted. Adjuvant nanoparticles do not deliver but stimulate related pathways, which will enhance the absorption of these antigens, and thus, immunity is achieved (Zhao et al., 2014). There are various types of nanoparticles that are in use. They can be natural as well as synthetic, so lots of kinds are present as their usage has become more common in preparation. Nanoliposomes, immunostimulant complexes (ISCOMs), virus-like particles (VLPs), etc., are a few examples that are found useful.
DNA vaccines are now newly used for the same purpose. The definition given by the Norwegian Biotechnology Advisory Board is “the intentional transfer of genetic material (DNA or RNA) to somatic cells for the purpose of influencing the immune system.” This definition differentiates it from gene therapy (Hølvold et al., 2014). There are significantly less number of DNA vaccines in fish, but examples such as vaccines against IHNV and VHSV have shown promise at experimental levels (Evensen and Leong, 2013). With time one can expect even more DNA vaccines as well as RNA vaccines. Polypeptides as vaccines are already in use widely, so there is an adequate chance that RNA vaccines will find success in the near future. The attribute of the DNA vaccine to induce all the arms of the immune system makes it very promising. Unlike conventional viral vaccines, DNA vaccines may conserve the structure and antigenicity of transgenic particles or proteins (Heppell et al., 1998). They show excellent efficiency when given in early life stages (Corbeil et al., 2000). They provide immunity in large ranges of temperatures, they are easy to produce, and processes for most are nearly the same (Lorenzen et al., 2009). As their replication is not a difficult task, modifications are not a tough task (Hansen et al., 1991). DNA plasmids cannot multiply in fishes but show great results and can be used at doses 100 times lower than in other animals (Embregts and Forlenza, 2016). Nucleic acids closely mimic antigens during intracellular pathogenic infection.
Bio-encapsulation in cases of oral vaccination for larva feed such as artemia and rotifers has been attempted, which has been successful. The amount of vaccine given is also in this way regu- lated as larvae do not consume a large amount of feed (Kawai et al., 1989). Microencapsulation of vaccines in polymers such as liposomes, PLGA, and alginates is very useful as they protect the antigen from the digestive tract (Tafalla et al., 2013).
In most cases, live vaccines are more effective as they mimic natural pathogenic infections.
Modern ways include subunits, recombinant particles, and DNA or RNA particles as vaccine con- stituents. Immunization has enhanced economic potential for farmers in aquaculture. Antibiotics and therapeutic drugs have been used against diseases, but resistance to medication, overuse, etc., are the concerns. A sudden reduction in the use of antibiotics in Norwegian Salmon after starting immunization has been achieved because this method is sustainable and gives beneficial results (Ma et al., 2019). In Norway, the yearly usage of antibiotics has reduced from 47 tons to 1 ton (Lillehaug et al., 2003). Viral outbreaks such as koi herpesvirus disease (KHVD), infectious salmon anemia virus (ISAV), viral hemorrhagic septicemia (VHS), and bacterial outbreaks such as mycobacteriosis and disease caused by Piscirickettsiasalmonis have been identified as the primary reason for the destruction of cultures (Dadar et al., 2019).
Inactivated vaccines are produced by culturing pathogenic organisms, and formalin is used to kill microbes without affecting the protective immunity. The preparation cost of viral vaccines is very high, and the effect is weaker than anticipated. Some fish viruses do not grow well in the culture, so their preparation is tough (Dadar et al., 2019). Attenuated vaccines are being produced against diseases like KHV (Dhar et al., 2014). These undergo various scrutiny before being allowed for use on the farmer level. If the conventional ones do not produce good immunity, then-novel technologies create more potent vaccines. Genetically modified organisms have also been implied, though their release into the environment can be concerning. Synthetic peptide vaccines are sub- unit vaccines used for diseases like IHNV, rhabdovirus, birnavirus, etc. (Estepa et al., 1999). To enhance the performance of vaccines, various adjuvants are used to improve vaccine efficiency.
Nanoparticles have acted as adjuvants as they increase the surface area and generate quantum size effects. Nanoparticles deliver antigens to cells, and adjuvants will activate specific pathways
94 Fish Vaccines facilitating antigen uptake (Zhao et al., 2014). Virus-like particles do not contain infective nucleic acid but still have the structure for inducing immunity (Vinay et al., 2016).
6.4.2 lImItatIons
Immunization is a revolution in aquaculture, but improper methods have caused mishaps. Careful immunization after knowing wholly about the fish species’ immune system is of primary impor- tance. Vaccines are available only for considerable species, so people must choose carefully. In non- encapsulated oral fish vaccines, the protection was for a short duration which started to decrease after 2–3 months after vaccination (Embregts and Forlenza, 2016). In cases of oral vaccination, gut nature presents more challenges. Carnivorous fishes have more protease usage, and non-carnivorous fishes have more amylase activity to digest plant matter. Nanoparticles have given more options to prepare vaccines but come with some limitations. As they can cross the blood-brain barrier (BBB), their application is challenging (Yildirimer et al., 2011). They also have proven to be toxic, and the estimation of toxicity is difficult. Its result ranges from cell necrosis to reactive oxygen spe- cies (ROS), causing apoptosis (Elsaesser and Howard, 2012). This effect also can reach humans and harm them. Vaccines give protection and adjuvants also have some side effects in fish, such as inflammation, pigmentation, intra-abdominal adhesion, etc. Injecting fish means causing stress, and spinal deformities have been noticed as side effects (Midtlyng et al., 1996).