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.