Progress 09/01/05 to 08/31/08
Outputs
OUTPUTS: Sub project titles are listed under Outcomes. (1) We have obtained 318 bacterial isolates from the trout GI tract and were able to re-grow and screen 238 of these for their ability to inhibit growth of F. psychrophilum under various conditions. One of these appears highly promising as a candidate probiotic for inhibiting Coldwater Disease. (2) Test results suggest that some F. psychrophilum proteins that are reactive with post-infection serum may be involved in Coldwater Disease pathogenesis and may serve as vaccine candidate antigens. (3) We successfully adapted a monoclonal antibody to detect F. psychrophilum in an ELISA format and determined the analytic sensitivity for kidney tissue. We also conjugated a fluorophore to the antibody and developed and validated a filter-membrane fluorescent antibody test for ovarian samples. This assay is not strictly quantitative, but it can be used to rank samples based on relative fluorescence. (4) Removing coelomic fluid from eggs of sub-fertile female rainbow trout before in vitro fertilization was shown to influence subsequent embryo viability. Tests indicate that while the coelomic fluid may contribute to the sub-fertile condition, an unknown factor(s) is associated with some eggs in batches from sub-fertile females and that is preventing their successful development. (5), Between 2005 and 2007, 47 chemistries were screened for efficacy in replacing carbaryl for control of burrowing shrimp. Based on efficacy, ease of application, cost, and registration potential, one compound, imidacloprid, was selected as a target for additional work. Between 2006 and 2008, imidacloprid efficacy was evaluated as a function of rate, season and method of application, timing, sediment type and vegetation coverage. Under most conditions, imidacloprid provided reasonable efficacy at 0.5 lbs ai/ac. Rates higher than that were required when conditions were not favorable, such as thick eelgrass cover or compacted soil. (6) With the help of personnel at the Hagermann Fish Culture Experiment Station, have completed growth trials of juvenile androgenetic rainbow trout. Our results are encouraging, indicating a significant difference in growth rate between the control strain and the High Growth rainbow strains. Production of clonal lines from the homozygous diploids has proven to be problematic. As an alternative strategy, a collaborator identified a growth QTL in an alternative cross of existing clonal lines. (7) Data were obtained challenging current dogma that suggest rainbow trout should be fed a low level of SBM during early feeding to improve use of higher levels of SBM during grow-out. Additionally, although probiotics appeared to improve soybean meal utilization during first feeding of SBM to rainbow trout, they provided only limited benefits once feeding was discontinued. (8) Mapping IHNV resistance loci in a rainbow X cutthroat has now been completed and published. We are also developing a map and testing for resistance loci in the OSU X Arlee clonal rainbow trout line cross. Additional work is also being done to study QTLs and map loci related to gene expression differences that may be relevant to disease resistance. PARTICIPANTS: Scientists from Washington State University include: Dr. Doug Call, Dr. Kevin Snekvik, Dr. Michael Dodson, Dr. Shulin Chen, Dr. Kim Patten, Mr. James Durfey, Dr. B. Dan Rodgers, Dr. Gary Thorgaard, and Dr. Ruth Phillips. Scientists from the University of Idaho include: Dr. Ken Cain, Dr. Rodney Hill, Dr. D.A.J. Stone, Dr. Wendy Sealey, Dr. Ken Overturf, Dr. Jim Liou, Dr. James Nagler, Dr. Barry Robison, Dr. David Stone, and Dr. Ron Hardy. Salaries for many graduate students, postdocs, and undergraduate students were paid by this grant. TARGET AUDIENCES: The target audience for the research results of this grant is the harvested-fish industry in Washington and Idaho. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
(1) Development of autochthonous probiotics to control disease outbreaks in aquaculture: contributed to training of four students and a technician. (2) Comparative genomics and proteomics of Flavobacterium psychrophilum: Moving toward vaccine development: a number of graduate students, postdoc research fellows, and support staff at UI and WSU have been supported, mentored, and trained in relation to this project. Patent application has been filed. (3) Development of a quantitative ELISA to detect Flavobacterium psychrophilum for broodstock management: This project produced a published Masters thesis anddirect dissemination of information to the Idaho Aquaculture industry. Results were also used to leverage a new grant to extend our efforts by further validation and assessment of the potential for applying the ELISA or FAT for disease management in hatcheries. (4) Assessing embryo mortality in rainbow trout: Removal of coelomic fluid from rainbow trout eggs before fertilization resulted in increased numbers of surviving embryos in egg batches from sub-fertile females. (5) Integrated development of alternative management tactics against burrowing shrimp on commercial oyster grounds: Field and laboratory experiments were conducted over a 4-year period to develop, assess and implement alternative controls for burrowing shrimp. These activities were aligned with numerous outreach events. Three burrowing shrimp grower/researcher workshops; four burrowing shrimp research sessions at the Pacific Coast Shellfish Grower Association annual meetings. (6) QTL analysis of growth rate in rainbow trout: Using the QTL cross generated by Dr. Drew, 14 growth QTL have been identified. The QTL identified were for 4 different growth parameters: length, weight, condition factor and specific growth rate. (7) Can Supplementing Starter Diets with Probiotics Increase Soybean Meal (SBM) Incorporation Levels in Practical Grow-Out Diets for Rainbow Trout: At the request of the United States Soybean Board (who wrote a letter of support for this proposal), a presentation was made as part of the Plant Proteins in Aquafeed Workshop. The study design and objectives were also presented to the Capital Hill Lunch and Learn Series in Washington DC. These approaches were also included as a framework to address plant products in aquatic species by Plant Products in Aquafeeds Working Groups. Three additional presentations have also been conducted. (8) Genetic control of IHNV resistance and growth in rainbow trout:
Publications
- Sealey, W.M., F.T. Barrows, C.E. Smith and R. Hardy. 2008. (Invited). Dietary strategies to optimize production of rainbow trout fed plant-based diets. UNJR Bilateral Meeting. Yokohama, Japan.
- Sealey, W.M., 2007. (Invited). Probiotics As A Means to Increase Plant-Based Ingredient Utilization in Rainbow Trout. USTF Annual Meeting. San Antonio, TX.
- Sealey, W.M., F.T. Barrows, E. Herman and S. LaPatra. 2007. Probiotics Increase Soybean Meal Tolerance Levels In Practical Grow-out Diets for Rainbow Trout. World Aquaculture 2007, San Antonio, TX.
- Shah, D.H., Cain, K.D., Wiens, G.D., and Call D.R. 2009. Challenges associated with heterologous expression of Flavobacterium psychophilum proteins in Escherichia coli. Marine Biotechnology.
- Plant, K.P., LaPatra, S.E., and Cain, K.D. 2009. Vaccination of rainbow trout (Oncorhynchus mykiss) with recombinant and DNA vaccines produced to Flavobacterium psychrophilum heat shock proteins 60 and 70. Journal of Fish Diseases 32(6):521-34
- LaFrentz, B.R., LaPatra, S.E., Call, D.R.., and Cain, K.D. 2008. Development and characterization of rifampicin resistant Flavobacterium psychrophilum strains and their potential as live attenuated vaccine candidates. Vaccine 26: 5582-5589.
- Chen, J., Davis, M.A., LaPatra, S.E., Cain, K.D., Snekvik, K.R., and Call, D.R. 2008. Genetic diversity of Flavobacterium psychrophilum recovered from commercially raised rainbow trout Oncorhynchus mykiss (Walbaum) and spawning Coho salmon Oncorhynchus kisucth. Journal of Fish Diseases 31: 765-773.
- Cain, K.D., and LaFrentz, B.R. 2007. Laboratory Maintenance of Flavobacterium psychrophilum and Flavobacterium columnare. Current Protocols in Microbiology (Book Chapter) 6:13B.1.1-13B.1.12.
- LaFrentz, B. R., Lindstrom, N. M., LaPatra, S. E., Call, D. R., and Cain, K. D. 2007. Electrophoretic and Western blot analyses of the lipopolysaccharide and glycocalyx of Flavobacterium psychrophilum. Fish and Shellfish Immunology 23, 770-780
- Ramsrud, A., LaFrentz, S. A., LaFrentz, B. R., Cain, K. D., and Call, D. R. 2007. Differentiating 16S rRNA alleles of Flavobacterium psychrophilum using a simple PCR assay. Journal of Fish Diseases, 30, 175-180
- Sudheesh, P. S., LaFrentz, B. R., Call, D. R., Seims, W. F., LaPatra, S. E., Wiens, G. D., and Cain, K. D. 2007. Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and a non-virulent strain of the fish pathogen Flavobacterium psychrophilum. Diseases of Aquatic Organisms, 74, 37-47
- Soule, M., LaFrentz, S., Cain, K., LaPatra, S., and Call, D.R. 2005. Polymorphisms in 16s rRNA genes of Flavobacterium psychrophilum correlate with elastin hydrolysis and tetracycline resistance. Diseases of Aquatic Organisms 65, 209-216.
- Soule, M., Cain, K., LaFrentz, S., and Call, D.R. 2005. Combining suppression hybridization and microarrays to map the intra-specific phylogeny of Flavobacterium psychrophilum. Infection and Immunity 73(6), 3799-3802.
- Lindstrom, NM, DR Call, ML House, CM Moffitt, and KD Cain. 2009. A quantitative enzyme-linked immunosorbent assay (ELISA) and filtration-based fluorescent antibody test as potential tools for screening Flavobacterium psychrophilum in broodstock. Journal of Aquatic Animal Health
- Hugunin, Heidi, James Parsons, and James J. Nagler (2008) The influence of coelomic fluid on in vitro fertilization success in rainbow trout (Oncorhynchus mykiss). Aquaculture 281:155-157.
- K. Patten, J. Durfey, J. Raskauskas, S Stern. 2005. An assessment of chemical controls and their methods of application for the management of burrowing shrimp in Willapa Bay. Pacific Coast Shellfish Grower Association Conference (abstract)
- Booth, S.R. and A. Suhrbier. 2006. Impact of alternative compounds applied for burrowing shrimp on benthic infauna. PCSGA / NSA Annual Conference (abstract).
- K. Patten, D. Aasen, J. Durfey, C. Hilley & Spikewheel Co. 2006. Design and evaluate subsurface chemical delivery systems and deep penetrating harrow for management of burrowing shrimp populations. Pacific Coast Shellfish Grower Association Conference (abstract)
- J. Durfey, C. Hilley, K. Patten. 2005. Subsurface shanking as a new technique for managing burrowing shrimp population in Willapa Bay. Pacific Coast Shellfish Grower Association Conference (abstract)
- K. Patten, D. Aasen, & D. Versteegen. 2007. Advances in chemical control of burrowing shrimp Pacific Coast Shellfish Grower Association Conference (abstract)
- Booth, S.R. 2007. Impact of experimental burrowing shrimp compounds on the benthic infauna. Pacific Coast Shellfish Grower Association Conference (abstract)
- Patten, K. 2007. Design and evaluate subsurface chemical delivery systems and deep penetrating harrow for management of burrowing shrimp populations. Coast Shellfish Grower Association Conference abstract)
- Hemberry, Christina. 2008. A soil modification to mitigate burrowing shrimp on commercial oyster grounds. MS Thesis, Civil Engineering University of Idaho.
- K. Patten, D. Aasen. 2008. Efficacy and non-target impacts of imidacloprid for burrowing shrimp control. Pacific Coast Shellfish Grower Association Conference (abstract)
- Sealey, W.M., F.T. Barrows, C. E. Smith, K. Overturf, and S.E. LaPatra 2009. Soybean Meal Level And Probiotics In First Feeding Fry Diets Alter The Ability Of Rainbow Trout Oncorhynchus mykiss To Utilize High Levels Of Soybean Meal During Grow-Out. Aquaculture Nutrition. (submitted)
- Barroso, R.M., P.A. Wheeler, S.E. LaPatra, R.E. Drew and G. H. Thorgaard, 2008. QTL for IHNV resistance and growth identified in a rainbow (Oncorhynchus mykiss) X Yellowstone cutthroat (Oncorhynchus clarki bouvieri) trout cross. Aquaculture 277: 156-163.
- Bernard D., B. Riteau, J.D. Hansen, R.B. Phillips, F. Michel, P. Boudinot, A.J. Benmansour. 2006. Costimulatory receptors in a teleost fish: typical CD28, elusive CTLA4. J. Immunology 176:4191-4200.
- Laing, K.J., J.J. Zou, L.J. Purcell, R.B. Phillips, C.J. Secombs, and J. D. Hansen. 2006. Evolution of the CD4 family: teleosts possess two divergent forms of CD4 in addition to LAG3. Journal of Immunology 177:3939-3951.
- Landis, E.D., Y. Palti, J. J. DeKoning, R.B. Phillips, and J. D. Hansen. 2006. Mapping and functional genomics of the TAPBP and TAPBPR genes. Immunogenetics 58:56-59.
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Progress 09/01/06 to 08/31/07
Outputs
Eight open reading frames were identified from a draft sequence of the F. psychrophilum genome containing 5 putative antigenic proteins associated with the outer membrane plus a putative metallopeptidase, hemolysin and another virulence associated protein. Expression and purification are being optimized for vaccine trials. Recombinant expression and purification of HSP 60, 70 and an ATP synthase homolog are underway. A functional sandwich ELISA developed with monoclonal antibodies is specific, sensitive and functional with fish tissue samples. A flow-through trout culture system was modified for partial recirculation. Design and installation, evaluation of the water quality and carrying capacity, and conducting a production performance evaluation and cost analysis have been achieved. A method employing stratified flow was developed for several hydraulic conditions utilizing a lower brine layer to remove debris from commercial raceways. The feeding of soybean meal to
trout with or without the addition of probiotics was not efficacious. An assay is being developed which tests the changes in trout gut flora with the use of probiotics. An analysis has revealed two interval regions on the genetic linkage map of rainbow trout that are significantly associated with resistance to IHNV. Mapping of known genes related to disease resistance has revealed trout CD4 and LAG-3. In an analysis of 52 progeny (doubled haploid hybrid between OSU and Arlee), it was revealed that significant QTL-influencing log transformed cortisol levels (correlated with stress) were detected on two linkage groups. In subfertile rainbow trout families, the majority of early embryonic death occurred within the first 12 hours. Utilizing a 16,000 gene microarray to identify genes showing a variation in mRNA, an analysis identified 104 genes consistently showing significantly higher mRNA levels in the subfertile egg pools. Candidate genes have been selected for further analysis. The
genomic clones for all four rainbow trout myostatin genes have been isolated and characterized. Although satellite cells were successfully isolated from rainbow trout, over time few cells became activated and reentered the cell cycle even when cultures were exposed to IGF, FGF, GH and other growth factors and hormones. Washing trout with chlorous acid, acidified chlorine dioxide, peroxyacetic acid, nisin or lactic acid controls the growth of microbes. Nisin is the most effective treatment at 3 degrees C. Treatments with tartaric acid, fumaric acid and a proprietary peroxyacetic acid compound were the most effective for Listeria control at 7 degrees C. Mechanical and chemical control of burrowing shrimp populations reveal that the most efficacious control is achieved with Sevin (currently used) or Admire (imidacloprid) when injected into the tidal flat with a spikewheel device.
Impacts
Progress has been made on all objectives of this project. Protective antigens are being identified and expressed which will lead to a vaccine for Cold Water Disease. Monoclonal antibodies, an ELISA for the detection of F. psychrophilum, and a microarray will facilitate detection of the bacterium that causes large losses of juveniles in rainbow trout hatcheries. Genetic mapping of genes related to IHNV resistance and immune competence will be important for breeding. Work on embryonic death will aid the trout egg industry in improving the germplasm that is shipped worldwide. Major strides are being made in preserving trout muscle and roe that will enhance the palatability, safety and value of aquaculture products. Work is underway which will validate the use of Admire for the control of burrowing shrimp in oyster cultures.
Publications
- 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.
- Swan C.M. 2006. Identification of a Localized Mucosal Immune Response in Rainbow Trout (Oncorhynchus mykiss) and Characterization of Immunoglobulin M and Other Proteins from Serum and Mucus. University of Idaho, M.S. Thesis.
- Stoddard, J., Parsons J.E., and Nagler J.J. 2005. Early Onset of Embryonic Mortality in Sub-Fertile Families of Rainbow Trout (Oncorhynchus mykiss). Reprod. Fert. Develop. 17: 785-790.
- Shin, J. H., Kang, D. H., and Rasco B. A. 2006. Survival and Growth of Listeria Monocytogenes in MAP Packaged Fresh Rainbow Trout. World Aquaculture 37(3): 6-10.
- Rasco B.A. and Bledsoe G. E. 2006. Surimi and Surimi Analogs. Handbook of Food Sciences and Technology. Marcel Dekker. NY NY. Chapter 160. pp. 1-7.
- Al-Holy, M. A. and Rasco, B. A. 2006. Characterization of Salmon (Oncorhynchus keta) and Sturgeon (Ascipenser transmontanous) Caviar Proteins. J. Food Biochem. 30: 422-428.
- Lin, M., Mousavi, M., Al Holy, M., Cavinato A. G., and Rasco, B. A. 2005. Rapid Near Infrared Spectroscopic Method for the Detection of Spoilage in Rainbow Trout (Oncorhynchus mykiss) Fillet. J. Food Science 71(1):S 018-023.
- Al-Holy, M., Wang, Y., Tang, J. and Rasco, B. 2004. Dielectric Properties of Salmon (Oncorhynchus keta) and Sturgeon (Ascipenser transmontanus) Caviar at 27 Mhz and 915 Mhz as a Function of Temperature. Food Research International 70(4):564-570.
- Lucas, M.D., R. E. Drew, P. A. Wheeler, P.A. Verell, and G. H. Thorgaard. 2004. Behavioral Differences among Rainbow Trout Clonal Lines. Behavior Genetics 34: 355-365.
- Laing, K.J., J. J. Zou, L.J. Purcell, R. B. Phillips, C. J. Secombes, and J. D. Hansen. 2006. Evolution of the CD4 Family: Teleosts Possess Two Divergent Forms of CD4 in Addition to LAG3. Journal of Immunology 177:3939-3951.
- Landis, E.D., Y. Palti, J. J. DeKoning, R. B. Phillips, and J. D. Hansen. 2006. Mapping and Functional Genomics of the Tapbp and Tapbpr Genes. Immunogenetics 58:56-59.
- Bernard, D., Riteau, B., Hansen, J.D., Phillips, R. B., Michel, F., Boudinot P., Benmansour, A. 2006. Costimulatory Receptors in a Teleost Fish: Typical CD 28, Elusive CTLA4. J. Immunology 176:4191-4200.
- Thorgaard G. H., K. M. Nichols, and R. B. Phillips. 2006. Comparative Gene and QTL Mapping in Aquaculture Species. Israeli J. Aquaculture 58:341-346.
- Garikipati, D., Ghar, S. A., and Rodgers, B.D. 2006. Identification, characterization and quantitative expression analysis of rainbow trout myostatin-1a and 1b genes. J. Endocrinol. 190(3):879-888.
- Kerr, T., Roalson, E. H., and Rodgers, B.D. 2005. Phylogenetic analysis of the myostatin gene sub-family and the differential expression of a novel member in zebrafish. Evo Devo 7(5) 391-401.
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Progress 09/01/05 to 08/31/06
Outputs
The O-polysaccharide of the F. Psychrophilum lipopolysaccharide is being tested to determine its protective effect. 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. It was discovered with suppression subtraction hybridization techniques and mixed genome microarrays that Flavobacterium psychrophilum can be divided into two distinct phylogenetic lineages. Ten important markers were discovered that identify the trout lineage, but which are absent from the salmon lineage. 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. 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. The 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).
Identification of transcriptional regulatory elements within the rainbow trout myostatin gene promoters and their relationship to growth of trout has just begun. One of the new objectives for this project is the isolation of authochthonous probiotics as antagonistic to Flavobacterium psychrophilum. A number of candidate bacteria have been isolated from the intestine of rainbow trout and are being tested for their ability to inhibit F. psychropilum in culture. Sensory profile analysis, on- going microbiological testing, assessment of storage stability, and water phase salt measurements have been performed to validate product for the new sturgeon caviar industry in Southern Idaho. A number of alternative pesticides to carbaryl have been tested to control the burrowing shrimp. Products with greatest efficacy are zeta-cypermethrin, crushed chrysanthemum, and other extracted natural pyrethrins. New equipment was tested for the subsurface injection of chemicals into the tidal flat to
control the shrimp. The mean vertical distribution of shrimp under various tidal or diurnal conditions was discovered to be 25 cm.
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. A new sturgeon caviar industry is being developed in Southern Idaho based on work by scientists/extension educators at WSU and the University of
Idaho.
Publications
- 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. Polymorphisms in 16s RNA genes of Flavobacterium psychrophilum correlate with elastin hydrolysis and tetracycline resistance. 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.
- 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. Chapter 159. Seafood products Science and technology . Handbook of Food Science. Marcel Dekker, NY NY.
- Bledsoe, GE. and Rasco, BA 2005. Chapter 161. Caviar and fish roe. Handbook of Food Science and Technology. Marcel Dekker, NY NY.
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