Protecting the Health of Farmed Fish
David R. Nelson, Professor
Department of Biochemistry, Microbiology, and Molecular Genetics
Aquaculture plays an ever-increasing role in
meet ing both national and global demands for seafood. Although it is estimated
that 70 percent of the world fish stocks are fully exploited or depleted, our
appetite for fish and other seafood is projected to increase from 72 million
metric tons (in 1993) to at least 91 million metric tons by 2010. To satisfy
the demands of an increasing population with an increasing taste for seafood,
the UN Food and Agriculture Organization (FAO) has projected that aquaculture
production must be doubled. Unfortunately, fish, like other animals, get diseases
when crowded together. As we become more reliant upon farmed fish, we will have
to develop newer, faster, and more accurate methods to detect, prevent, and
treat infectious disease agents (viruses, bacteria, and parasites) of fish.
My lab has been engaged in this effort for several years. We are interested
in a particularly virulent fish disease known as vibriosis. Vibriosis, one of
the major bacterial diseases affecting fish, bivalves, and crustaceans, is caused
by the bacterium Vibrio anguillarum (see Fig. 1). The distribution of
vibriosis is worldwide and causes great economic loss to the aquaculture industry:
It is estimated that the annual losses of cultured fish in Japan alone exceed
$30 million. Vibriosis is often the major limiting factor in the successful
rearing of salmonids.
V. anguillarum typically causes a hemorrhagic septicemia. Infected fish display skin discoloration and red patches around the base of the fins, vent, and mouth; the gastrointestinal tract and rectum become distended and filled with fluid; and necrotic lesions form in the abdominal muscle. Infected fish become lethargic and suffer heavy mortalities ranging from 30 percent to 100 percent. It appears that most infections with V. anguillarum begin with the colonization of the fish gastrointestinal tract. The bacteria are strongly attracted to intestinal mucus. Once they have colonized the fish intestine, they appear to penetrate the intestinal wall and cause a systemic infection resulting in disease and death.
While there are vaccines against vibriosis, they are somewhat crude preparations and protect only against specific serotypes or strains of the organism. Thus, if the vaccine manufacturer guesses incorrectly about the V. anguillarum strains that are likely to be present in a given area, the vaccinated fish will not be protected and fish farmers could lose entire fish crops. We are attempting to develop more efficacious vaccines through our studies of V. anguillarum . We have been studying the interaction of the bacteria with salmon intestinal mucus in order to determine 1) whether V. anguillarum cells can grow in mucus, 2) whether growth in mucus induces (turns on) specific genes and proteins in the bacteria, 3) whether any of the proteins produced during growth in mucus can be used as vaccines against infection, and 4) whether V. anguillarum mutants lacking the ability to grow in mucus could serve as live vaccines.
During the last few years I have found, with the graduate and undergraduate students in my lab, that V. anguillarum cells grow explosively in mucus. The mucus environment appears to provide the cells with all the nutritional requirements necessary to grow at their maximum rate. This finding makes it clear that colonization of the intestine allows the invading bacterial pathogen to amplify rapidly prior to infection and disease. Thus, the fish host is infected with a massive, overwhelming dose of bacteria---resistance is futile.
During growth in mucus, V. anguillarum cells express a number of new proteins. These proteins are not present during growth in other media. Some of these proteins are on the surface of the bacterial cells and appear to be ideal candidates for the development of new vaccines. Several surface proteins are specifically induced by growth in mucus and appear to be responsible for the influx of nutrients into the bacterial cell. Some of these proteins have been purified for further study. We are working to clone the genes that encode for these proteins with the goal of using the recombinant proteins as vaccines against vibriosis.
A protease is another protein whose expression is greatly increased during growth in mucus. Proteases are proteins that degrade other proteins and are often excreted into the surrounding medium. The V. anguillarum protease is excreted and may be a factor in enhancing the virulence of this organism. The protease is specifically induced by exposure of the cells to fish intestinal mucus (see Fig. 2). It is important to determine how mucus enhances the expression of the gene that encodes the protease. Finally, we have constructed and identified a mutant strain of V. anguillarum that is unable to grow on mucus. This strain of bacterium appears to have a mutation in a single gene (see Fig. 3). The absence of a functioning copy of this gene prevents mutant growth. Previously, we hypothesized that strains unable to grow in mucus should be unable to cause disease in fish. We tested this hypothesis by infecting salmon with either the mutant or its virulent parent strain. The parent strain caused disease and death in fish when inoculated at doses as small as 1,000 bacteria per fish. The mutant failed to cause disease at doses as large as 100 million bacteria per fish. Our hypothesis was correct: a mutant strain of V. anguillarum unable to grow in mucus was avirulent---unable to cause disease. Could our avirulent mutant strain also protect fish against infection by a virulent strain? We tested this question by inoculating fish previously vaccinated with our avirulent strain with lethal doses (100,000 or 1,000,000 bacteria per fish) of a virulent V. anguillarum. As usual, we included a control group of fish that had not been vaccinated. The control fish were also challenged with a lethal dose of virulent bacteria. All our control fish died within 3 days of receiving the lethal dose of bacteria. By contrast, none of the vaccinated fish that were inoculated with the lethal dose of virulent bacteria died or showed any signs of illness over the 14 days that the observations were made. Our hypothesis is correct: strains of V. anguillarum unable to grow in mucus are avirulent, and these avirulent strains are suitable as live vaccine strains.
Despite this success, there is much more to do. We plan to examine the regulation of the mucus-induced genes. How does mucus induce the expression of specific genes? What in the mucus do the bacteria use as nutrients? In conjunction with other faculty at URI, we have also identified another species of vibrio, Vibrio carchariae, that causes peritonitis in summer flounder. This organism also grows explosively in intestinal mucus. What are the similarities, and what are the differences between V. anguillarum and V. carchariae? Do different vibrio species have different or similar systems to regulate gene expression in mucus? Can we develop specific rapid tests to detect the presence of these pathogens in the water column or in the fish prior to an outbreak of disease? Can we develop protective and economical vaccines that will allow the development and expansion of the aquaculture industry?
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