Progress 09/15/02 to 09/14/05
Outputs
The O-polysaccharide of the F. Psychrophilum lipopolysaccharide is being tested to determine its protective effect. Other antigenic proteins are being identified by 2-dimensional electrophoresis. Mass spectrometry has identified two of the proteins as heat shock proteins HSP 60 and HSP 70. The respective genes have been cloned into expression vectors for testing as vaccines. We have validated a functional sandwich ELISA, based on monoclonal antibodies, for detecting F. psychrophilum. The assay is both specific and sensitive and will function in the presence of fish kidney and ovarian tissue. Basic Immunology: With respect to objectives on elucidating mucosal immunity to F. psychrophilum in trout, it has been found that localized antigen specific antibody is produced within the mucosal tissues, but there appears to be a restricted ability to respond to antigen delivered to mucosal sites, i.e., the gut. Success has been achieved in assembling and optimizing a multiple
tank indoor rearing facilities including trickling filters for biofiltration and fine solids removal, a moving bead filter and a new baffle settling basin to remove large particles. Another project is evaluating a partial recirculating system incorporating an airlift pump to profitably raise trout. Two new baffle configurations, both hinged and moving have been designed to address flow velocity limitations and improve waste transport in the rearing areas of raceways. With regard to genomic analyses of accelerated growth rates in rainbow trout, androgenetic diploids have been produced as the basis of creation of clonal lines of trout with high growth rates. These animals will form the basis of genetic analysis of growth rate in industry related strains. Already the High growth (Troutlodge and Donaldson strains) have shown significant differences in growth from the control strain. With respect to mapping the genetic regions for IHNV resistance and growth in rainbow trout, substantial
progress has been made. The IHNV challenge trials have been completed and the QTL analysis of markers related to IHNV disease resistance is underway. Analysis of 402 markers has resulted in the identification of 45 linkage groups with a total map length of 3767 cM and 155 total markers mapped. A genetic region associated with IHNV resistance has been identified. With regard to determining the number and effects of QTLs for variation in stress responsiveness and test for associations with relevant genes, stress response was analyzed in three inbred lines. The Arlee line (long time domesticated line) has a reduced stress as compared to SW (another longtime domesticated lines) and OSU lines. Since SW has also been domesticated for a period of time, this result goes against the hypothesis that domestication reduces the stress (as measured by elevated cortisol levels). Two salmonid DNA microarrays (3,500 and 16,000 genes Center for Biomedical Research, University of Victoria BC) have been
employed to analyze the DNA from eggs of sub-fertile and fertile trout. Six different genes show abundance patterns that differ more than two fold between batches of eggs that are designated as fertile or sub-fertile.
Impacts
This work is producing diagnostics based on microarrays and ELISAs that will lead to more efficient detection of disease in rainbow trout aquaculture. Studies of the immunology of trout will lead to knowledge of how the trout processes disease antigens and achieves immunity. Development of vaccines for the most important pathogens of the industry will make the trout aquaculture industry more profitable. New aquaculture engineering techniques will make water reuse systems more economical and will render traditional flow-through aquaculture facilities more environmentally friendly. Genetic studies will allow breeding schemes which produce high growth, stress tolerant and disease resistant fish. Identification and culling of low fertility females will allow trout spawning operations to become more economical and efficient.
Publications
- Hansen J.D. Landis, E. G. Phillips, R.B. 2005. Discovery of a new Ig heaviy chain isotype in rainbow trout : Implications for a novel B cell developmental pathway in teleost fish. Proceedings of the National Academy of Sciences 102:6919-6924.
- Al-Holy, M. Lin, M. and Rasco, A. 2005. Destruction of Listeria monocytogenes in sturgeon (Acipenser transmontanus) caviar using a combination of nisin with chemical antimicrobials or moderate heat. J. Food Protection. 68(3):512-520.
- Rasco, BA and Bledsoe, GE. 2005. CH 160.Surimi and surimi analogs. Handbook of Food Science and Technology. Marcel Dekker, NY NY. In press.
- Rasco, BA and Bledsoe, GE. 2005. Chapter 159. Seafood products Science and technology . Handbook of Food Science. Marcel Dekker, NY NY.
- Soule, M., K. Cain, S. LaFrentz, D.R. Call. 2005. Combining suppression subtractive hybridization and microarrays to map the intraspecies phylogeny of Flavobacterium psychrophilum. Infection and Immunity 73:3799-3802.
- Soule, M., S. LaFrentz, K. Cain S. LaPatra, and D.R. Call. 2005. Polymornphisms in 16s RNA genes of Flavobacterium psychrophilum correlate with elastin hydrolysis and tetracycline resustance. Diseases of Aquatic Organisms 65: 209-216.
- True, B. J. Johnson B., and S. Chen 2004. Reducing phosphorous discharge from flow-through aquaculture facilities. I. Facility and Effluent Characterization. Aquacultural Engineering. 32:129-144.
- True, B.J. Johnson G. and S Chen, 2004. Reducing phosphorous discharge from flow-through aquaculture facilities II. Hinged and moving baffle designs to improve waste transport. Aquacultural Engineering 32:125-160.
- True, B. J. Johnson, B. and S. Chen. 2004. Reducing Phonsporous discharge from flow-through aquaculture facilities-III. Assessing high rate filtration media for effluent solids and phosphorus removal. Aquacultural Engineering, 32:161-170.
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Progress 01/01/04 to 12/31/04
Outputs
New products from trout: Pasteurization and in-package pasteurization of whole trout and processed trout containing products (caviar, center-of-the plate entrees) by steaming methods, radiofrequency and microwave methods were evaluated. Salt and lipid content played a large role in the heat transfer in the products. Bone content did not affect the dielectric heating in the foods. A novel tube-heating device was constructed which may be useful in processing extruded food from minced muscle tissues. Cold Water Disease: Immunization experiments using crude extracts of LPS (O-Antigens and Core) were ineffective. There is continued focus on characterizing the higher molecular weight components of this material and characterization of this material with proteomic methodologies. DNA Microarray Detector for Bacterial Kidney Disease (BKD): The PCR microarray assay for R. salmoninarum was compared with the polyclonal ELISA for sensitivity and specificity. To date, the ELISA
remains the method of choice for detection of BKD organisms. Genetic control of IHNV resistance in trout: Substantial variation in expression of IHNV resistance has been noted between the various clonal lines of trout. Work on the QTLs associated with natural killer cell-like activity in rainbow trout has been completed. There is one QTL that is associated with this important innate immune activity. A new filter was developed for the recirculating system.
Impacts
New safe and palatable products with added value will make the trout industry more profitable. Studies of the immune system of trout, diagnostic methods which identify the presence of disease organisms early, and the development of vaccines against the prime diseases plaguing the industry will eventually make management practices in the industry more reasonable and efficient. New aquaculture engineering techniques will make water reuse more economical and render traditional flow-through aquaculture facilities more environmentally friendly.
Publications
- LaFrentz, B.R., LaPatra, S.E., Jones, G.R., and Cain, K.D. 2004. Protective immunity in rainbow trout Oncorhynchus mykiss following immunization with distinct molecular mass fractions isolated from Flavobacterium psychrophilum. Diseases of Aquatic Organisms 59, 17-26.
- Al-Holy, M., Quinde, A., Guan, D., Tang, J. and Rasco, B. 2004. Thermal inactivation of Listeria innocua in salmon (Oncorhynchus keta) caviar using conventional glass and novel aluminum TDT tubes. J. Food Protection. 67(2):383-386.
- Lin, M, Al-Holy, M, Al-Qadiri, H, Kang, DH, Cavinato, A.G. and Rasco, B.A. 2004. Discrimination of intact and injured species of Listeria spp. by Fourier transform infrared spectroscopy and principal component analysis. J. Agric. Food Chem. 52:5769-5772.
- Lin, M., Al-Holy, M., Mousavi-Hesary, M., Al-Qidiri, H., Cavinato, A.G. and Rasco, B.A. 2004. Rapid and quantitative detection of the microbial spoilage in chicken breast muscle by short-wavelength near-infrared (SW-NIR) Spectroscopy. J. Applied and Environmental Micro. 39:148-155.
- Ohta, Y., Landis, E., Boulay, T., Phillips, R.B., Collet, B., Secombes, C.J., Flajnik, M. F., and Hansen, J.D. 2004. Homologs of CD83 from elasmobranch and teleost fish. J. Immunology 173:4553-4560.
- Gonzalez, SF, MJ Krug, ME Nielson, Y. Santos, and DR Call. 2004. Simultaneous detection of marine fish pathogens using multiplexed PCR and DNA microarrays. Journal of Clinical Microbiology 42:1414-1419.
- Zimmerman A.M., Evenhuis J.P., Thorgaard GH, Ristow S.S. 2004. A single major chromosomal region controls natural killer cell-like activity in Rainbow Trout. Immunogenetics 55: 825-835.
- Chen S. Reducing phosphorous discharge from flow-through aquaculture: II: Hinged and moving baffles to improve waste transport. 2004. Aquaculture Engineering, 32:145-160.
- Stoddard, J., Parsons, J.E., and Nagler, J.J. (2004) Rapid onset of early embryonic mortality in sub-fertile families of rainbow trout (Oncorhynchus mykiss). Aquaculture (CALL NAGLER)
- Straub, TM, Quinonez Diaz, MD, CO Valdez, DR Call, and DP Chandler. 2004. Using DNA microarrays to detect multiple pathogen threats in water. Water Science and Technology: Water Supply 4:107-114.
- Warsen, A.E., M.J. Krug, S. LaFrentz, D.R. Stanek, F.J. Loge, and D.R. Call. 2004. Simultaneous discrimination between 15 fish pathogens using 16S rDNA PCR and DNA microarrays. Appl. Environ. Microbiol. 70:4216-4221.
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Progress 01/01/03 to 12/31/03
Outputs
A number of lots of doubled haploid trout were raised in order to map the resistance against the IHNV and for mapping genes associated with growth rates. Considerable genes included MHCIa, MHCIb, ABCB2B, ABCB3B (2 loci) TCR alpha, TCR beta, CD83, tapasin, Nramp alpha and IgL. A major discovery was that the entire MHCI region with approximately 24 genes is completely duplicated in trout. With regard to examination of the innate immune of the trout, natural-killer like activity has been mapped to a single QTL. This activity is unlinked to fragmented MHC class I and class II regions, the leukocyte receptor cluster, the natural killer cell enhancement factor gene, the RAG-1 gene and two genes associated with resistance to IPNV. The importance of antibody protection against Flavobacterium psychrophilum (cold water disease) was established and allowed the immunoreactive antigens from a virulent strain of the pathogen to be characterized. Antibodies from fish showing near
and complete protection were directed at a 50kDa protein proteinase K resistant protein and at the O antigen of the pathogen. Genomic DNA has been extracted from a virulent and nonvirulent strains of F. psychrophilum in preparation for genomic subtraction experiments to locate the virulence factors associated with the pathogenic strain. A mixed strain microarray based on important Flavobacterium species is under construction. A microarray-based assay for the presence of Renibacterium salmoninarum has been developed which is sensitive down to 100 bacterial genomes. In the area aquaculture engineering, a novel moving baffle system was developed for moving fish waste in raceways and introduced to industry. Models of incipient motion of fish waste and food in raceways are in development. The aquaculture potential of the canals, laterals and drains and wasteways of the Columbia Basin project were assessed and it was concluded that much potential exists for aquaculture in these areas from
March to October each year. A newly designed recirculating cold water system was set up to produce large sized fingerlings during winter months. Stable isotopes of carbon and nitrogen have been used to determine the dynamics of carbon and nitrogen budgets as a function of development. It was found that C and N turnover may be potentially disrupted by environmental stressors. Fish may be fed food enriched with these stable isotopes and the isotopes can be used as a non-invasive tag, which can be measured in fin clips. In terms of high valued food products from aquacultured trout, new smoked products are being developed with a newly constructed microwave process at 915 MHZ. Salmon caviar has been successfully pasteurized using a tube heater system. A fried noodle containing 10-20% trout mince was produced and was well received in sensory tests. It was also found that the off-flavors in trout are caused by lipid oxidation and that this condition can be mitigated by better husbandry and
processing practices.
Impacts
This work is producing diagnostics based on microarrays which will lead to more efficient means to detect disease in trout culture. Identification of antigens, which can be used to immunize trout, will lead to new vaccines for devastating pathogens such as the causative agent of cold water disease. It is possible that the newly engineered baffle system will help reduce effluents from hatchery operations and that the new incipient motion analysis will reduce phosphorous in the effluent from hatchery operations. The new high valued products from trout muscle and roe will lead to more profit for the industry.
Publications
- Phillips RB, Zimmerman A, Noakes, M, Palti Y, Morasch M, Eiben L, Ristow S, Thorgaard G, Hansen JD. 2003. Physical and genetic mapping of the rainbow trout major histocompatibility regions: evidence for the duplication of the class I region. Immunogenetics 55:561-569.
- LaFrentz BR, LaPatra SE, Jones GR, Congleton JL, Sun B, Cain KD. 2002. Characterization of serum and mucosal antibody responses and relative percent survival in rainbow trout (Oncorhynchus mykiss) following immunization and challenge with Flavobacterium pyschropilum. Journal of Fish Disease 25:703-713.
- Lafentz B, LaPatra S, Jones G, Cain K. 2003. Passive immunization of rainbow trout (Oncorhynchus mykiss) against flavobacterium psychrophilum, the causative agents of coldwater disease and rainbow trout fry syndrome. Journal of Fish Diseases 26:377-384.
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Progress 01/01/02 to 12/31/02
Outputs
Progress has been made toward (1) producing high value food products from aquacultured trout, (2) elucidating natural immunity in trout, (3) examining the immune response of trout to Flavobacterium psychrophilum, (4) preparation of DNA from F. Psychrophilum (5) construction of a F. psychrophilum genomic library (6) design of integrated aquaculture/agriculture systems including filters and establishing pilot systems for demonstration and extension and (7) the design of a new biofilter and solids removal strategy for recirculating trout culture. Preliminary experiments were conducted with salmonid caviar using microwave processes. A radiofrequency processing unit circulating water of a particular ionic strength around containers during processing was shown to reduce the come-up time for thermal processing by a factor of four and produce a firmer textured product. RF pasteurization experiments were conducted on salmonid caviar to kill Listeria monocytogenes. Dielectric
properties of trout muscle and roe containing different levels of salt were estimated. Lipid oxidation measurements for minced trout products point to the nature of off-flavors in those preparations. We have established a genetic linkage map for the trout based on the OSU X HC cross. By mapping with microsatellites, we have established the syntenic relationship of the OSU X HC map and the OSU X Arlee map. A number of immune-related genes were placed on the map including nonspecific cytotoxic cell activity, natural killer cell enhancement factor, and Class I and Class II major histocompatibility antigens. NCC activity maps as a single QTL. F. pyschrophilum, antigens recognized by serum antibodies from immunized trout include numerous antigens in the 70-200kD region. A challenge experiment has indicated that protective capacity was found mainly in the high molecular weight region and the protective activity was attributed to a 50 kDa polysaccharide now being characterized. Genomic DNA
has been extracted from the CSF 259-93 strain and the ATCC 49418 strain of F. psychrophilum in preparation for the subtractive library. In the subproject integrating aquaculture and agriculture, there has been information gathering for the development of the integrated system configuration and the associated management strategies. Different options have been provided for rearing fish in their pre-irrigation seasons. Some test trials were conducted and valuable lessons were learned about coordination between aquaculture operations and the local irrigation districts. Concerning the new biofilter and solids removal strategy for recirculating trout aquaculture, a design has been produced for a vertical settling tank, and a laboratory system has been designed and constructed which utilizes a two-stage solids removal process utilizing a double drain for efficiency. There has been assembly of an additional experimental system allowing evaluation of the nitrification efficiency of the new
filtration system and the interactions between nitrification and solids removal.
Impacts
We are developing high valued muscle and roe products which, if accepted by the public, will make the trout industry more profitable. Knowledge concerning the innate and specific immune systems of trout will be valuable for the development of vaccines against pathogens devastating trout in aquaculture. Extensive study of the immune responses to F. psychrophilum will lead to new vaccine strategies against this economically important pathogen. Extension activities will play a major role in educating the public about emerging production systems which create a symbiotic system utilizing aquaculture and agriculture resources more efficiently and economically.
Publications
- No publications reported this period
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