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United States Department of Agriculture

Agricultural Research Service

2006 Annual Report

1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
Rainbow trout is an important aquaculture fish species both for its commercial value as a food fish as well as for its recreational value to fishermen in the northern United States and Canada. A major handicap of large-scale culturing of this fish species is disease outbreaks resulting from infection by bacterial, fungal, parasitic and viral pathogens. Development of strains of rainbow trout resistant to pathogen infection via genetic manipulation will be the solution to this problem. To resolve this problem we have initiated work to produce new rainbow trout strains with increased resistance to infection by bacteria, viruses, fungi and parasites by genetic engineering. Furthermore, we have also conducted studies to characterize genes that are responsive to infection by pathogens in rainbow trout in order to develop alternative strategies of disease protection in rainbow trout.

The overall goals for the entire project are:

(1) Develop rainbow trout brood stocks with disease resistant genetic trait for aquaculture. (2) Identify and characterize the genes responsive to induction by cecropin B in rainbow trout macrophage cells. (3) Develop rainbow trout brood stock with enhanced muscle growth for aquaculture by down-regulating MSTN gene.

2.List by year the currently approved milestones (indicators of research progress)
Milestones for FY2005

1. Conduct challenge studies of F2 Rbt-99 fish with a viral pathogen, infectious hematopoietic necrosis virus (IHNV). 2. Characterize F2 transgenic fish with respect to transgene inheritance and transgene expression. 3. Continue to breed fish from F2 Rbt-99 transgenic fish to homozygosity for the cecropin gene. 4. Continue screening genes in trout macrophage cells that are regulated by antimicrobial peptides. 5. Produce RNAi gene constructs and test the feasibility of these constructs in down regulating MSTN-1 gene in a rainbow trout cell line in vitro.

Milestones for FY2006

1. Conduct challenge studies of F3 Rbt-99 and F2 Rbt-02 fish against A. salmonicida and IHNV. 2. Initiate breeding of Rbt-99 transgenic fish to homozygous by androgenesis. 3. Using microarray technology complete screening of genes that are regulated by antimicrobial peptide. 4. Complete characterization of cecropin resistant bacterial pathogens for infectivity to hosts. 5. Cryopreserve sperm of F2 Rbt-02 fish. 6. Identify strong muscle-specific promoters from rainbow trout. 7. Determine whether antimicrobial peptide can serve as an adjuvant in vaccination.

Milestones for FY2007

1. F4 Rbt-99 challenge studies. 2. Breed Rbt02 to homozygous. 3. Cryopreserve F3 Rbt-99 sperm and F2-Rbt-02 sperm 4. Complete the genotyping of homozygous Rbt99 fish. 5. Constructing and testing RNAi for MSTN 6. Breeding homozygous Rbt-02 fish 7. Confirm microarray analysis results by real-time PCR 8. Publish results of research with Rbt99 transgenic fish.

Milestones for FY2008

1. Conduct scale-up challenge studies with homozygous Rbt99 fish. 2. Conduct F3 Rbt-02 challenge studies. 3. Cryopreservation of F3 Rbt-02 sperm. 4. Genotyping homozygous Rbt-02 fish. 5. Produce P1 transgenic fish carrying RNAi for MSTN.

Milestones for FY2009

1. Genotyping P1 transgenic fish carrying RNAi for MSTN. 2. Conduct scale-up challenge studies with homozygous Rbt-02 fish 3. Cryopreserve sperm samples of homozygous Rbt-99 and Rbt-02 fish 4. Comparing the expression patterns of immune responsive genes in transgenic fish and non-transgenic fish.

Milestones for FY2010

1. Establish F1 family of transgenic fish carrying RNAi of MSTN. 2. Re-confirm the results of scale-up challenge studies with newly established animals by crossing homozgous transgenic fish (i.e., Rbt-99 and Rbt-02) with non-transgenic fish. 3. Prepare manuscripts reporting results of challenge studies with Rbt02 fish; and the expression patterns of immune responsive genes in transgenic fish and non-transgenic fish.

4a.List the single most significant research accomplishment during FY 2006.
Another anti-microbial peptide found that is induced in rainbow trout. We have shown for the first time that another antimicrobial peptide, hepcidin, in rainbow trout could be induced by not only bacterial infection but also by synthetic poly-inosinic-poly-cytidylic acid (poly-I:C), a mimic of viral double-stranded (ds) RNA. The induction of hepcidin in fish by bacterial and viral infections may be important in protecting the animal from secondary bacterial infection by either directly killing the bacteria or restraining free iron level in the serum.

4b.List other significant research accomplishment(s), if any.
Cecropin transgenic fish exhibit resistance to a viral pathogen IHNV: We have confirmed that two subfamilies of S7 founder family (i.e., S7#342-F695 and S2#375-F180), two subfamilies of S8 founder family (S8#505-G275 and S8#505-G231), four subfamilies of S9 founder family (S9#638-F297, S9#659-F073, S9#746-F509 and S9#659-541) are resistant to A. salmonicida and IHNV. We have also confirmed that another four families Rbt-99B founder fish are resistant to IHNV.

Some bacterial pathogens may develop resistant to antimicrobial peptides: We have demonstrated conclusively that some bacterial pathogens can gain resistance to antimicrobial peptide via physiological adaptation, but will return to non-resistant status if the selection pressure is removed. Furthermore, the resistant pathogens do not exhibit higher pathogenicity than the non-resistant counterparts.

4c.List significant activities that support special target populations.

4d.Progress report.
This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the University of Connecticut. Additional details of research can be found in the report for the parent CRIS 1930-31000-007, Utilizing Genetics for Enhancing Cool and Cold Water Aquaculture Production.

5.Describe the major accomplishments to date and their predicted or actual impact.
Annual loss of fish as a consequence of infection by bacterial and viral pathogens impacts the productivity of aquaculture of trout and other finfish/shellfish tremendously in the United States. There is an urgent need to develop strategies to overcome this problem. Through our earlier studies, it became clear that manipulation of antimicrobial peptide genes in trout may result in development of new strains of fish that are resistant to bacterial and viral infection. We have demonstrated that the gene products of cecropin and CF-17 genes can effectively kill fish bacterial and viral pathogens. By introducing cecropin and CF-17 genes into rainbow trout embryos, we have produced transgenic trout that exhibited strong resistance to bacterial and viral pathogens. In addition, we have obtained evidence indicating that antimicrobial peptides will not result in selecting microorganisms with resistance characteristics to these peptides. Work is underway to breed rainbow trout with homozygous antimicrobial peptide genes for large-scale grow out. Once the work is completed, the first potential impact of this project is that the brood stocks of these fish could be used by trout farmers for commercialization without the worry of disease outbreaks. The second impact of the project is that the same strategy can be adopted to develop highly disease resistant finfish and crustacean species for aquaculture. With the availability of these transgenic fish, the U.S. could become the leader in providing disease-resistance fish stocks for aquaculture throughout the world.

6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?

7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Chiou, P Peter, Bols, Niels, Douglas, Sue and Chen, Thomas T. 2005. Regulation of Immune-Relevant Genes in the Trout Macrophage Cell Line RTS-11 by Antimicrobial Peptides. Developmental and Comparative Immunology 30: 797-806.

Last Modified: 5/5/2015
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