Linda Benedict, Tiersch, Terrence R., Cooper, Richard K. | 5/8/2009 7:29:28 PM
Richard K. Cooper and Terrence R. Tiersch
About 10 percent of the annual channel catfish crop is lost to infectious diseases. The most important of these diseases are Edwardsiella ictaluri, Flavobacterium columnare and channel catfish virus. Only the antibiotics Romet and oxytetracycline, which can be incorporated into feed, are approved by the Food and Drug Administration (FDA) to treat E. ictaluri and F.columnare, and there is no treatment for channel catfish virus. The farmer has to rely on good management to prevent outbreaks of disease. Even the most carefully managed pond can experience disease problems, however.
With treatment options limited, much emphasis in the past several years has been placed on vaccine development, with the most progress to date being made on E. ictaluri and channel catfish virus. Although vaccines hold promise, the drawback is that a vaccine has to be made for each disease-causing organism, and each has to be approved by the USDA. The entire process takes several years.
Several years ago, we began to investigate other ways to protect catfish against these diseases. Our goal is to produce a fish that alone can withstand infection or have its immune system further strengthened with vaccines to specific pathogens as the vaccines become available.
To accomplish the goal of enhancing the catfish immune system, we chose a gene for a lytic peptide that is controlled by an acute phase promoter, which can be compared to an on/off switch. Lytic peptides are small peptide molecules produced by a wide array of animals from invertebrates to mammals, including humans. There are many classes of lytic peptides based on their structure, but most share a common component. They are part of the first line of defense for an organism, and they kill pathogens by destroying the cell membrane and releasing cellular components. Lytic peptides range from moderately lytic, in which they kill only specific types of bacteria and infected cells, to the very lytic. Melittin, for example, is the major component of honeybee venom. We chose the lytic peptide cecropin B from the giant silk moth, Hyalophora cecropia, because it kills a wide range of Gram negative and Gram positive bacteria without damaging host cells. Previous work had shown cecropin B to be very effective in killing E. ictaluri, one of the primary pathogens against which we wanted to protect channel catfish.
Another key component of the cecropin B gene is the acute phase promoter (APR) that controls expression of the peptide. A promoter controls when a gene will be “turned on.” There are basically two types of promoters: those that are expressed, or on, at some level all the time, and those that are turned on only when needed. The APR promoter is “off” when it is not needed (when no pathogen is present), but it can be turned on quickly by molecules in the immune system that are responding to an invasion by a pathogen. This system allows the catfish to express the cecropin B peptide when a pathogen is encountered and not expend unneeded energy when there is no pathogen.
To deliver the cecropin B gene to the catfish genome, we developed a system to optimize DNA incorporation (see page 25). Using a technique called electroporation, in which a small electrical charge is applied to the cells that momentarily creates pores in the cell membrane to allow the DNA to enter the cell, we transferred the plasmid vector containing the cecropin B gene to unfertilized channel catfish eggs. The eggs were fertilized using artificial spawning techniques developed in our laboratory, hatched and allowed to grow in a recirculating tank system housed in a laboratory approved for transgenic research.
When the fish resulting from the experiment were large enough to handle, a small sample of blood was taken from each fish and analyzed for the presence of the cecropin B gene using a technique called the polymerase chain reaction. More than half of the fingerlings were positive for the cecropin gene.
We have demonstrated, too, that the APR promoter functions in the manner intended. None of the cecropin B product is detectable in healthy, transgenic fish, but it is detectable 12 hours after the fish has been exposed to E. ictaluri.
To demonstrate whether or not cecropin B could protect transgenic catfish against E. ictaluri, we exposed an equal number of transgenic and nontransgenic fish with a virulent strain of the bacterium. Three days after infection, the fish were cultured for E. ictaluri. The non-transgenic, or control fish, had very high levels of bacteria in the hind kidney; in the transgenic fish, 50 percent of the fish had no detectable levels of bacteria, 34 percent had greatly reduced levels and the remaining 14 percent were no different from the non-transgenic fish. The differences in protection against E. ictaluri may be caused by the number of copies of the cecropin gene that inserted into the catfish chromosome. More copies could mean higher levels of lytic peptide resulting in more rapid clearance of the bacterium. We have shown that the use of gene transfer in channel catfish is feasible and that protection against E. ictaluri is possible.
Our long-term goal is to provide the catfish industry of Louisiana with a superior strain of channel catfish that has the ability to fight infection against the common diseases now plaguing the industry. This can be accomplished with an animal carrying a gene such as cecropin B and live attenuated vaccines as they become available. One key feature of having these animals available, however, is to produce a fish that is sterile. Recently, we have begun to pursue avenues to induce sterility (see page 16).
Funding support came from the USDA and the Louisiana Catfish Promotion and Research Board. Personnel involved included Brandye Smith, Jan Lousteau, Jackie McManus, Mark Bates, Greg Roppolo, Quiyang Zhang, Herman Poleo and Gang Yu. Richard K. Cooper, Associate Professor, Department of Veterinary Science, and Terrence R. Tiersch, Professor, Aquaculture Research Station, LSU Agricultural Center, Baton Rouge, La