Vaccines In Aquaculture Research Articles

Immunostimulants: The best alternative to vaccination and antibiotics in aquaculture

Immunostimulants comprise a group of biological and synthetic compounds that enhance the non-specific cellular and humoral defense mechanism in animals. These substances such as levamisole and glucan, peptidoglycon, chitin, chitosan, yeast and vitamin combinations as well as various products derived from plants and animals are effective in prevention of diseases. Use of expensive chemotherapeutants and antibiotics for controlling disease have widely been criticized for their negative impact like residual accumulation in the tissue, development of the drug resistance and immunosuppression, thus resulting in reduced consumer preference for food fish treated with antibiotics (Anderson,1992). Hence, instead of chemotherapeutic agents, increasing attention is being paid to the use of immunostimulants for disease control measures in aquaculture. The use of immunostimulants for the prevention of fish diseases is considered an attractive and promising approach. These immunostimulants mainly promote the function of phagocytes and increase their microbicidal power. They also enhance production of acute phase proteins that trigger the innate defence mechanism of the host. However, a controversy exists over the use of these substances, as in a few cases they have failed to render enhanced protection or increase in immunity. Long-term exposure, over and under dosing problems, timing and method of application of these substances are discussed here. This review article mainly considers the recent developments in this area to control fish diseases describing all different types and sources of immunostimulants, their characteristics, objective and evaluation of the mode of work.Key words: Immunostimulants, disease control, alternative, fish, antibiotics, aquaculture

Extremophile extracts and enhancement techniques show promise for the development of a live vaccine against Flavobacterium columnare

The effects of temperature, ionic strength, and new cryopreservatives derived from polar ice bacteria were investigated to help accelerate the development of economical, live attenuated vaccines for aquaculture. Extracts of the extremophile Gelidibacter algens functioned very well as part of a lyophilization cryoprotectant formulation in a 15-week storage trial. The bacterial extract and trehalose additives resulted in significantly higher colony counts of columnaris bacteria ( Flavobacterium columnare) compared to nonfat milk or physiological saline at all time points measured. The bacterial extract combined with trehalose appeared to enhance the relative efficiency of recovery and growth potential of columnaris in flask culture compared to saline, nonfat milk, or trehalose-only controls. Pre-lyophilization temperature treatments significantly affected F. columnare survival following rehydration. A 30-min exposure at 0 °C resulted in a 10-fold increase in bacterial survival following rehydration compared to mid-range temperature treatments. The brief 30 and 35 °C pre-lyophilization exposures appeared to be detrimental to the rehydration survival of the bacteria. The survival of F. columnare through the lyophilization process was also strongly affected by changes in ionic strength of the bacterial suspension. Changes in rehydration constituents were also found to be important in promoting increased survival and growth. As the sodium chloride concentration increased, the viability of rehydrated F. columnare decreased.

Do uncertainty analyses reveal uncertainties? Using the introduction of DNA vaccines to aquaculture as a case

The Walker and Harremoës (W&H) uncertainty framework is a tool to systematically identify scientific uncertainty. We applied the W&H uncertainty framework to elicit scientists' judgements of potential sources of uncertainty associated with the use of DNA vaccination in aquaculture. DNA vaccination is considered a promising solution to combat pathological fish diseases. There is, however, lack of knowledge regarding its ecological and social implications. Our findings indicate that scientists are open and aware of a number of uncertainties associated with DNA vaccination e.g. with regard to immune response, degradation and distribution of the DNA plasmid after injection and environmental release, and consider most of these uncertainties to be adequately reduced through more research. We proceed to discuss our experience of using the W&H uncertainty framework. Some challenges related to the application of the framework were recognised. This was especially related to the respondents' unfamiliarity with the concepts used and their lack of experience in discussing qualitative aspects of uncertainties. As we see it, the W&H framework should be considered as a useful tool to stimulate reflection on uncertainty and an important first step in a more extensive process of including and properly dealing with uncertainties in science and policymaking.

DNA vaccination in aquaculture — Expert judgments of impacts on environment and fish health

In this expert elicitation twelve Norwegian scientists were interviewed about environmental and health consequences of DNA vaccination in aquaculture. The intention was to elicit scientists’ judgments of potential sources of uncertainty associated with the use of DNA vaccines. Emphasis was put on three incidents: i) immune response following the DNA vaccination, ii) tissue distribution and expression of the injected plasmid DNA (pDNA) and iii) environmental release of DNA vaccines. We present what the experts identified as potential consequences of introducing DNA vaccines to aquaculture. Our findings support the need for more research on the stability of the pDNA, unintended immunological impacts and integration of the pDNA in the genome of the recipient organism. The study suggests that expert elicitation can improve communication of scientific uncertainty and thereby advance the scientific discussion. We argue that our findings support the importance of including several experts in the process of risk assessment and decision support, as different scientists express diverging opinions of the consequences involved.

Biotecnología y vacunas de ADN para animales acuáticos

Biotechnology has been used extensively in the development of vaccines for aquaculture. Modern molecular methods such as polymerase chain reaction (PCR), cloning and microarray analysis have facilitated antigen discovery, construction of novel candidate vaccines, and assessments of vaccine efficacy, mode of action, and host response. This review focuses on DNA vaccines for finfish to illustrate biotechnology applications in this field. Although DNA vaccines for fish rhabdoviruses continue to show the highest efficacy, DNA vaccines for several other viral and bacterial fish pathogens have now been proven to provide significant protection against pathogen challenge. Studies of the fish rhabdovirus DNA vaccines have elucidated factors that affect DNA vaccine efficacy as well as the nature of the fish innate and adaptive immune responses to DNA vaccines. As tools for managing aquatic animal disease emergencies, DNA vaccines have advantages in speed, flexibility, and safety, and one fish DNA vaccine has been licensed.

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass ( Lates calcarifer) to protect from Vibrio ( Listonella) anguillarum

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass ( Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.

Expression and Immunogenicity Analysis of Two Iron-regulated Outer Membrane Proteins of <italic>Vibrio parahaemolyticus</italic>

Genes of two iron-regulated outer membrane proteins of Vibrio parahaemolyticus zj2003, a pathogenic strain isolated from large yellow croaker (Pseudosciaena crocea), psuA and pvuA, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli BL(21)(DE(3)). The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of the proteins, groups of large yellow croaker were immunized with the purified recombinant psuA, pvuA or both, by intraperitoneal injection. Antibody response was assessed by enzyme-linked immunosorbent assay. Titers to the recombinant proteins increased from log(2) 3.25 to log(2) 9.80, 4-8 weeks following immunization. The relative percent survival of the groups vaccinated with psuA, pvuA, or a combination of the two, reached 50%, 62.5% and 75%, respectively. Western blot analysis was carried out with the serum from unvaccinated survival fish after infection. Both recombinant proteins were detected, indicating that these two proteins of V. parahaemolyticus zj2003 were immunogenic and could produce synergistic effects during in vivo infection, and they might be considered as important components for developing an aquaculture vaccine against this pathogen.

Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS). To identify antigens associated with virulence or host immunity, we compared total and immunogenic proteins of cellular and extracellular products (ECP) between a virulent (CSF-259-93) and non-virulent (ATCC 49418) strain of F. psychrophilum. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis of total cellular proteins revealed only minor differences between the strains; however, separation of ECP showed that proteins were differentially expressed. Western blot analysis using rainbow trout (Oncorhynchus mykiss) anti-CSF-259-93 sera showed greater reactivity to proteins of the virulent strain, including many > 50 kDa. Further analysis by 2-dimensional electrophoresis (2DE) identified numerous differences between the strains. Western blot analysis combined with 2DE identified several immunogenic proteins that reacted with the antisera and were shared between the 2 strains. However, at least 15 immunogenic proteins appeared to be unique to the virulent strain, while 4 such proteins were identified in the non-virulent strain; 8 proteins unique to the virulent strain and 6 shared proteins were further analyzed for identification by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Of these, 3 immunogenic proteins (heat shock proteins HSP 60 and HSP 70) and 2 other proteins (ATP synthase and thermolysin) were conclusively identified. The 2 highly immunogenic heat shock proteins were shown to share extensive homology with heat shock proteins of related bacteria. This approach for antigen identification may provide a basis for targeted vaccine development against CWD and RTFS.

Cloning, expression and immunogenicty analysis of five outer membrane proteins of Vibrio parahaemolyticus zj2003

Genes of five outer membrane proteins of Vibrio parahaemolyticus zj2003, including OmpW, OmpV, OmpK, OmpU and TolC, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of these proteins, large yellow croaker ( Pseudosciaena crocea) were immunized by intraperitoneal injection. Antibody response was assessed by method of enzyme-linked immunosorbent assay. Titres to all five recombinant proteins increased during 4 to 8 weeks post immunization, within the range of log 2 values of 5.75 to 10.8. Recorded relative survival percent (RPS) of the vaccinated groups varied from 80% to 90%, while 10 fish in control group all died. Western blot tests were undertaken with the serum of survival fish after experimental infection. Except for recombinant TolC, the other four recombinant proteins were recognized by the serum. It is indicated that four outer membrane proteins of V. parahaemolyticus zj2003, including OmpW, OmpV, OmpU and OmpK, are immunogenic during in vivo infection, which would be of some significance in developing efficient vaccine in aquaculture. This is the first report of successful vaccination against V. parahaemolyticus with purified recombinant outer membrane proteins.

Improved uptake of plant-derived LTB-linked proteins in carp gut and induction of specific humoral immune responses upon infeed delivery

Oral vaccination of fish is an effortless and stress free immunisation method which can be used for almost any age. However, vaccination via the mucosal route does have disadvantages. For example, the vaccine may induce tolerance and has to be protected to escape digestion. Also the vaccine should be efficiently delivered to immune-competent cells in the gut or other lymphoid organs. In addition, it should be cost effective. Here we present a novel fish vaccination model using potato tubers as vaccine production and delivery system. The model vaccines discussed here include fusion proteins consisting of a gut adhesion molecule (LTB) and a viral peptide or green fluorescent protein (GFP) expressed in potato tubers. The adhesion molecule mediates binding to and uptake from the gut, whereas the viral peptide or GFP functions as model vaccine antigen provoking the induction of an immune response. We demonstrate that fusion to LTB facilitates an elevated uptake of the model vaccines in carp gut mucosa. The plant-derived fusion proteins also elicit a specific systemic humoral immune response upon oral application of crude tuber material incorporated into a standard dietary feed pellet. The data presented here show the promising potentials of the plant as a production system for oral vaccines in aquaculture and feed mediated immunisation of fish.

Analysis of long-lived plasma cell production and regulation: Implications for vaccine design for aquaculture

The importance of long-lived plasma cells (LPC), over other antibody-secreting cells (ASC), in the generation of long-term immunity and memory in mammals has been the focus of considerable recent interest. Studies suggest that long-term immunity to infectious agents, such as viruses, may be primarily due to the expression of antibody from this unique population of ASC. Unfortunately due to the relative paucity of studies on teleost immune cells, virtually no information exists on LPC, or ASC diversity in these species. However, recent work in our laboratory has revealed that trout possess functionally diverse ASC populations, including plasmablasts, short-lived plasma cells and long-lived plasma cells. Importantly, as in mammals, these populations are not uniformly distributed among all immune tissues. This is of particular significance with respect to long-lived plasma cells, which appear to exclusively reside within, or migrate to, the anterior kidney. Previous teleost studies have operated under the assumption that all ASC are plasma cells. However, we have found that virtually the entire peripheral blood lymphocyte (PBL) ASC response is comprised of plasmablasts, while the trout plasma cell response resides within the anterior kidney. Thus, the assumption that the in vitro behavior of ASC from PBL is identical to that of plasma cells and that they are responsible for in vivo antibody responses, is flawed. Based on this information we propose an alternative model of functional differentiation and regional distribution of ASC in the trout and pose some important implications for the development of immunological memory and aquaculture vaccines based on this model.

Evaluation of eukaryotic promoters for the construction of DNA vaccines for aquaculture

We evaluated fish promoters as an alternative to viral promoters in the construction of DNA vaccines for aquaculture. A carp β-actin promoter drove expression of the luciferase gene in live fish tissue to levels comparable to the CMVtk promoter.

Viral vaccines for aquaculture

A description of the types of viral vaccines that are being developed for fish is presented in this review. All three types of vaccines, i.e. killed, attenuated, and subunit vaccines, have worked to some extent in fish under controlled laboratory conditions. However, with the exception of a killed vaccine for spring viremia of carp, there are no commercially available viral vaccines for fish. The introduction of these vaccines to aquaculture will require an understanding of immune recognition in fish and the development of cost-effective ways of producing a safe, immunogenic vaccine. A review of available information on vaccines for infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, spring viremia of carp virus, and channel catfish virus is presented. These are the main viruses that have been investigated for vaccine development. Very little information is available on vaccines for other viral diseases of fish.

Vaccines in aquaculture: The search for an efficient delivery system

The development of a strong aquaculture industry is dependent, in part, on advances in the diagnosis and treatment of fish diseases such as vibriosis. Vaccination is one method of lowering the risks associated with the occurrence of disease. The vaccination of large populations of marine animals, however, presents special problems and considerations. The delivery methods employed, for example, must be effective not only from the point of view of protection against infection but they must also be cost-effective. This review attempts to assess some of the currently employed vaccine delivery methods, as well as describing emerging technologies which may have application in the aquaculture industry.