Biotechnological Approaches to Disease Prevention in Aquaculture
Marta Gomez-Chiarri, Assistant Professor
Department of Fisheries, Animal, and Veterinary Science
Not only humans get sick. Fish and shellfish
fill our oceans and aquariums with color and provide us with a healthy and delicious
source of food. They also are afflicted with infectious diseases. These diseases
can have an enormous ecological, economic, and social impact, constraining the
expansion of fish and shellfish farming and endangering fisheries. For example,
diseases like Dermo (caused by the parasite Perkinsus marinus) and MSX
(caused by another parasite, Haplosporidium nelsoni) have decimated oyster
populations on the east coast. Wildlife managers, fly fishermen, and researchers
are concerned about the recent impact of whirling disease in wild trout. More
than 50 diseases affect fish and shellfish that are cultured in the United States,
causing losses of tens of millions of dollars annually.
Despite the impact that diseases have on aquatic organisms, we know relatively little about which pathogens affect fish and shellfish, how these organisms fight diseases, and what we can do to prevent and treat diseases. The use of drugs and antibiotics in aquaculture is highly regulated to avoid risks to public safety and to prevent the development of resistant strains of pathogens. Consequently, farmers are left with few resources other than the use of preventive measures such as early diagnosis, good husbandry techniques, vaccination, and the use of strains of fish and shellfish genetically resistant to diseases.
Biotechnological and molecular techniques have proven to be extremely successful in the biomedical field and could be equally useful in aquatic health. Biotechnology can be used to design specific and sensitive diagnostic tools, study the immune systems of fish and shellfish, and look at the relationship between pathogens and their hosts. The creation of disease-resistant strains with gene transfer and the development of efficacious vaccines and treatments and new ways to deliver them are also made possible with biotechnology. The goal of our research is to prevent diseases in fish and shellfish, using whatever research tools are available. Three different research projects being conducted in my laboratory illustrate different approaches to disease prevention and treatment. These are the identification of the organism responsible for a disease outbreak in summer flounder, the development of DNA vaccines for the prevention of bacterial diseases in finfish, and the evaluation of antimicrobial peptides in the treatment of shellfish diseases.
The first step in fighting infectious diseases is to know the enemy. For example, an outbreak of an unknown disease hit a summer flounder farm located in Quonset Point, Rhode Island last summer. The farmer was losing around 200 fish per day and because he lacked treatment, he felt helpless to stop the mortalities. Three research teams at URI (the laboratories of Jennifer Specker, David Nelson, and my group) worked together to identify the cause of the mortalities. We used a series of microbiological tools to isolate several potential pathogens and design a series of challenge experiments that determined which pathogen was causing the disease. Using biochemical tools (comparison of enzyme profiles with profiles from known bacterium) and molecular tools (comparison of a DNA sequence from our isolate with sequences in a genetic database) we identified the culprit as Vibrio carchariae, a bacterium originally isolated from skin lesions in sharks. We are in the process of designing diagnostic tools to detect the presence of the pathogen and vaccines to prevent further outbreaks.
The development of cheap and effective vaccines for the prevention of diseases caused by viruses, parasites, and intracellular bacteria in finfish has proven to be a difficult task. As a result, few commercial vaccines are available for innoculation against these kinds of diseases. Traditional vaccines used to prevent fish diseases consist of either killed pathogens (called bacterins in the case of bacteria) or attenuated versions of the pathogen (live vaccines). Other alternatives being investigated include the use of purified or genetically engineered antigens from the pathogen (recombinant vaccines) and DNA vaccines. Among these, DNA vaccines are considered the "third revolution" in vaccine development. In DNA vaccines, a gene coding for a protein from the pathogen is inserted into a bacterial plasmid (a small, circular DNA molecule that can be used as a vector) that contains all the elements necessary for the gene to be expressed in human (or fish) cells. These plasmids are replicated in bacterial culture and the DNA is purified and transferred to live fish by intramuscular injection. No one really knows how, but some cells in the fish pick up the DNA and express the protein. The immune system recognizes the protein as foreign and mounts a response against the antigen, which protects the fish when they eventually become infected with the pathogen. The main advantages of DNA vaccines are that they are simple to prepare and that DNA can be produced in large quantities with high purity. Furthermore, DNA is highly stable and resistant to temperature extremes, which facilitates the storage, transport, and distribution of vaccines. In addition to these commercial considerations for vaccine production and distribution, DNA vaccines also have immunological advantages. Since the immune system sees the expressed antigen in a way that is similar to how it sees virus and intracellular bacteria, DNA vaccines are especially useful in the prevention of diseases caused by these pathogens.
It has been shown in experimental models that DNA vaccines can provide protection from a variety of pathogens that cause diseases like hepatitis B and C, AIDS, tuberculosis, and malaria. DNA vaccines have also been tested in fish by several research groups (including ours) and have been shown to protect rainbow trout against infectious hematopoietic necrosis virus, viral hemorrhagic septicemia, and bacterial kidney disease, diseases that have a serious impact on salmonid aquaculture throughout the world. However, several problems remain to be solved before these vaccines can be used in the farms. Relatively little is known about how DNA vaccines work and how we can improve them. Also, there are safety issues that need to be addressed before vaccine companies can commercialize these vaccines. Our DNA vaccine research is being funded by Alpharma Inc., a company with years of experience in selling fish vaccines. We are developing, in close collaboration with researchers at Alpharma, safe plasmid constructs specially designed for the immunization of fish with DNA. These plasmid constructs will satisfy regulatory guidelines from the Food and Drug Administration, the U.S. Department of Agriculture, and the European Union. Instead of using sequences of viral origin to drive the expression of the foreign antigen, we are using sequences of fish origin, like b-actin promoter from carp. We have compared the activity of several of our constructs using in vivo assays in fish and have observed that they are at least as efficient at driving the expression of a foreign gene as the constructs that are used in the design of DNA vaccines for humans.
Because shellfish lack an antibody immune response, they canšt be vaccinated. This is one reason that we need to explore different strategies to prevent and treat infectious diseases in shellfish. Since the use of antibiotics is highly regulated, we decided to make use of the innate mechanisms that shellfish use to fight infections. A variety of strategies are employed by marine invertebrates to kill invasive or opportunistic microorganisms. These mechanisms include phagocyte-mediated killing, agglutination, encapsulation, and the release of microbicidal molecules. The latter category includes lysosomal enzymes, antiprotozoan agents, and antimicrobial peptides (AMPs). AMPs are attractive as potential agents for prevention and treatment of pathogen outbreaks in aquaculture systems. These peptides have potent antimicrobial, antiendotoxin, and, in some cases, antifungal properties. They play a crucial role in the defense of bacteria, plants, invertebrates, and vertebrates. AMPs are small molecules that interact directly with the microbial cell surface, forming holes that lead to the death of the microbes. Their special structure (they have a net positive charge) and the differences between the membranes of pathogens and hosts, enable the AMPs to destroy the pathogen without harming the host cell. Furthermore, the method by which these peptides work may make it more difficult for bacteria to develop resistance against AMPs. In collaboration with Lenore Martin, Assistant Professor of Biomedical Sciences at URI, we are testing the activity of different AMPs, like tachyplesin which is isolated from the horseshoe crab, against different pathogens that affect oysters in culture conditions similar to the marine environment. Our goal is to determine which AMPs are most efficient in the treatment of oyster diseases. One of the problems still to be resolved is the best and most cost-effective way to deliver AMPs to the oysters. One solution is to genetically engineer oysters by introducing the gene coding for the AMP into the germline to create strains with increased peptide production that would be more resistant to infections.
With the help of biotechnology, aquaculture will meet the growing demand for seafood and will produce healthy fish without the release of drugs and antibiotics into the environment. This, in turn, will relieve enormous pressure on marine fisheries and help preserve biodiversity in our oceans.
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