Progress 08/15/08 to 08/14/11
Outputs
OUTPUTS: 1. Genetic Control on IHNV Resistance and Growth in Rainbow Trout A major innate anti-viral defense mechanism is the type I interferon (IFN) system. Recent research shows that the system in trout and salmon consists of multiple gene families and duplicated genes within each family. IHNV is sensitive to IFN. We propose to identify all of the IFN genes so each one can be studied individually in response to the virus. Changes in expression of specific genes of interest are being analyzed for correlations with growth and/or disease resistance. Polymorphisms are being identified and the genes are being mapped to chromosome regions. 2. Typing and Mapping of the Rainbow Trout Genome Work has proceeded on identification of single nucleotide polymorphism (SNPs) from transcribed genes. When the analysis is completed we should have a significant number of new SNPs in immune genes that can be used to develop an SNP chip. Work has also proceeded on bioinformatically assembling bacterial artificial chromosomes (BACs) containing the four estrogen receptors in the rainbow trout. These parologous genes result from ancestral duplication of the rainbow trout genome. 3. Sex Determination in White Sturgeon Analysis has revealed the sturgeon genome to apparently contain a lower proportion of markers behaving as disomic (diploid) Mendelian segregating loci as contrasted with rainbow trout genetic profiles in mapping families and those in most vertebrates. Currently we have identified 101 mappable AFLP markers, and we are seeking 800-1000 markers to provide sufficient coverage of the linkage groups. Our initial efforts suggest a female heterogametic (ZW female, ZZ male) mechanism in this species, based on the presence of female- but not male-specific bands. 4. Strawberry Disease in Rainbow Trout We employed molecular methods (16s rDNA libraries) to identify bacteria present in SD lesions and identified a sequence from a Rickettsia-like-organism (RLO). Subsequent PCR testing showed a highly statistically significant association between the RLO and SD lesions. Collaborations with the University of Stirling (U.K.) also demonstrated that the RLO sequence identified from our work is found in fish afflicted with "red-mark syndrome" in the U.K. We are attempting to identify the environmental reservoir for the RLO. 5. Flavobacterium psychrophillum To date we have tested approximately ten different recombinant proteins, but we were unable to identify an antigen that can protect trout against injection challenge with our virulent strain. We are focusing remaining project resources to essential experiments needed for licensing an attenuated vaccine (B.17) recently developed by our group. This vaccine was patented by the University of Idaho in 2010 ("Vaccines for Diseases of Fish" issued on June 22, 2010 as US Patent No. 7,740,864) and results to-date have been very promising. The B.17 vaccine is currently being field tested. PARTICIPANTS: Gary Thorgaard, School of Biological Sciences, Washington State University (WSU)
Ruth Phillips, School of Biological Sciences, WSU
Amit Dhingra, Horticulture and Landscape Architecture, WSU
Douglas Call, Professor, College of Veterinary Medicine, WSU
Kevin Snekvik, co-investigator, Associate Professor, College of Veterinary Medicine, WSU
Devendra Shah, collaborator, Assistant Professor, College of Veterinary Medicine, WSU Ken Overturf, University of Idaho (UIdaho), National Fish Hatchery in Hagerman, ID
Ron Hardy, UIdaho, National Fish Hatchery
James Nagler, UIdaho
Barrie Robison, UIdaho
Ken Cain, co-investigator, Associate Professor, College of Natural Resources, Univ. Idaho, Moscow, ID
Gary Fornshell, Aquaculture Extension Specialist, College of Agriculture and Life Sciences, Univ. Idaho, Twin Falls, ID
Karen Plant, Postdoc, Hagerman Fish Culture Experiment Station, UIdaho In addition to the investigators listed above, the project involved the following research faculty and graduate students:
In the Gary Thorgaard WSU lab:
Mary May, graduate student
Joseph Brunelli, research assistant professor at WSU, was involved in interpretation of the results.
Kris Christensen, PhD student in Genetics and Cell Biology at WSU, worked on the project and is using a significant portion of his dissertation research. In the Ruth Phillips WSU lab:
Jenefer DeKoning, a postdoc at WSU-Vancouver, was involved in bioinformatic analyses
Sonja Lloyd, graduate student, College of Veterinary Medicine, WSU
Carla Schubiger, graduate and pathology resident, College of Veterinary Medicine
James Derringer, Postdoc, College of Veterinary Medicine, WSU
Karol Gliniewicz, graduate student, College of Veterinary Medicine, WSU At the University of Idaho (UIdaho):
Tim Cavileer, a Research Scientist I at UIdaho, contributed to lab work and analysis of results.
Sam Hunter, PhD student in the Bioinformatics and Computational Biology program at UIdaho is working on this project and will use a significant portion for his dissertation research. Scott LaPatra, collaborator, Clear Springs Foods in Buhl, ID. Bind Canyon Aquaranch, a company actively involved in sturgeon farming in the Twin Falls, Idaho area who provided the samples for the study. TARGET AUDIENCES: Companies involved in aquaculture of salmonid fishes and other finfishes. Researchers associated with breeding companies, government agencies and universities with an interest in animal breeding, disease mechanisms and resistance and conservation. Researchers and companies involved in breeding of fishes for aquaculture; researchers involved in genetic typing of salmonid fishes for conservation and management programs. Aquaculture companies involved in sturgeon aquaculture; researchers and agencies concerned with conservation and management of sturgeon populations. Aquaculturists and fish health specialists. This work has targeted the research community, aquaculture professionals, fish health/diagnostic laboratories, and companies interested in fish vaccine development. Work supported in part through this project has caused a change in knowledge by demonstrating that a vaccine is feasible and has provided important tools for improved diagnostics. Results have been disseminated through refereed publications, presentations at meetings, and popular press and news avenues. Partnerships with two companies (Immunoprecise Antibodies and Aquatic Life Sciences) have been established, resulting in commercialization of diagnostic tools and hopefully a licensed vaccine for coldwater disease. PROJECT MODIFICATIONS: We have shifted our focus more to evaluation of our B.17 vaccine and away from testing of recombinant proteins. This was due to a need to provide data for vaccine licensing tempered with the limited success of the recombinant vaccine trials.
Impacts
1. IHNV Resistance: Additional genetic markers are being developed with broad application to both breeding and conservation issues in salmonids. The complexity of disease response and challenges associated with breeding for disease resistance in fishes is being recognized. 2. Typing and Mapping: Completion of the SNP identification work in the next few months should allow the identified markers to be incorporated into a large "SNP chip" which is being developed under a grant proposal to Yniv Palti of USDA in cooperation with Illumina. This SNP chip should facilitate mapping and identification of QTLs (quantitative trait loci) in rainbow trout and other salmonids, which should ultimately improve breeding programs. 3. Sex Determination: When completed, it is possible that genetic markers will be identified which can be used by sturgeon farmers to cull out males and increase the profitability of sturgeon aquaculture. 4. Strawberry Disease: This project supported completion of a Ph.D. dissertation and delivery of 14 presentations (talks or posters) at local, national and international meetings including at trade association meetings, plus publication of two papers in peer-reviewed journals. 5. Flavobacterium psychrophillum: At the University of Idaho this project is currently providing partial support for a postdoctoral research fellow and has contributed to a M.S. project completed in December, 2010. At Washington State University this project has provided partial support for a postdoctoral fellow and a PhD student. Many publications have been attributed to this project and if all upcoming vaccine field trials for our attenuated vaccine show protection, we expect this to be licensed and available commercially within the next year. Earlier work also supported the commercialization of a monoclonal antibody (FL43) for diagnostics, and this (along with FAT and ELISA protocols) is currently available to hatcheries and diagnostic labs through Immunoprecise Laboratories (Victoria, BC Canada).
Publications
- 1. Sudheesh, P.S., LaFrentz, B.R., LaPatra, S.E., Call, D., and Cain, K.D. (2004) Differential proteomic analysis of virulence associated and immunoreactive antigens of the salmonid pathogen, Flavobacterium psychrophilum. AFS Annual Fish Health Section Meeting, Kearneysville, West Virginia, July 25-28.
- 49. Cain, K.D., and LaFrentz, B. 2004. Coldwater Disease Workshop. Presented to Hatchery personnel from the trout industry. June 2004. College of Southern Idaho Hatchery.
- 50. Cain KD and Call DR (2004) Comparative genomics and proteomics for identification of virulence and immunogenic antigens. Bacterial Coldwater Disease Workgroup, USGS Western Fisheries Research Center, Seattle, WA, 5 February.
- 55. LaFrentz, B.R., LaPatra, S.E., Jones, G.R. and Cain, K.D. (2004) An investigation into the protective nature of Flavobacterium psychrophilum lipopolysaccharide against Coldwater Disease. AFS Annual Fish Health Section Meeting, Kearneysville, West Virginia, July 25-28.
- 56. Lane, S. Cain KD and Call DR (2004). Development of monoclonal antibodies for detection of the fish pathogen Flavobacterium psychrophilum. 6th Annual College of Veterinary Medicine Research Symposium, Pullman, WA, 14 October.
- 57. Soule, LaFrentz, Oatley, Krug, LaFrentz, Cain and Call. 2004. Comparative genetics of virulent and avirulent strains of Flavobacterium psychrophilum, etiological agent for cold water disease in salmonids. 6th Annual College of Veterinary Medicine Research Symposium, Pullman, WA, 14 October.
- 58. Soule, LaFrentz, Oatley, Krug, LaFrentz, Cain and Call (2004) Comparative genetics of virulent and avirulent strains of Flavobacterium psychrophilum, etiological agent for cold water disease in salmonids. ASM Annual meeting, New Orleans, LA, May.
- 59. LaFrentz, B.R., and Cain, K.D., Coldwater Disease, Extension Bulletin, 2004. Western Regional Aquaculture Consortium (WRAC) 9 pgs.
- 60. Plant, K.P., LaPatra, S.E., Call, D.R., and Cain, K.D. 2011. Immunization of rainbow trout (Oncorhynchus mykiss) with Flavobacterium psychrophilum proteins elongation factor-Tu, SufB Fe-S assembly protein and ATP synthaseB. Journal of Fish Diseases 34, 247-250
- 2. Sudheesh, P. S., Benjamin LaFrentz, Scott E. Lapatra, Douglas Call and Kenneth D. Cain, 2004. Differential proteomic analysis of virulence associated and immunoreactive antigens of the salmonid pathogen, Flavobacterium psychrophilum. Poster presentation. CRB Meeting, June 23-24.
- 3. Cain KD and Call DR (2005) Comparative genomics and proteomics for identification of virulence and immunogenic antigens. Aquaculture, ID-WA Research Review, Moscow, ID, February.
- 4. Cain, KD, Sudheesh PS, LaPatra SE, Weins GD, LaFrentz BR and Call DR (2005) Identification and expression of an immuno-reactive heat shock protein from Flavobacterium psychrophilum. American Fisheries Society/Fish Health Section, Minneapolis, MN, 27-29 July.
- 5. Call, D.R. (2005) Detection of microbial biodiversity in environmental samples. Invited seminar for MicroPad, A European FP5 Program, Camerino, Italy, September 19-21.
- 6. Soule, LaFrentz, Oatley, Krug, Cain and Call (2004) Virulent and avirulent strains of Flavobacterium psychrophilum compared using suppression subtractive hybridization. AAAS Annual meeting, Seattle, WA.
- 7. Soule, M, Cain KD, LaFrentz S, and Call DR (2004) Virulent and avirulent Flavobacterium psychrophilum strains compared using suppression subtractive hybridization and microarrays. Bacterial Coldwater Disease Working Group, Seattle, WA, FLAVO meeting, Seattle, 8 Sept.
- 8. Soule, M, LaFrentz S, Oatley M, Krug M, LaFrentz B, Cain KD and Call DR (2004) Comparative genetics of virulent and avirulent strains of Flavobacterium psychrophilum, etiological agent for coldwater disease in salmonids. WSU Center for Integrated Biotechnology Retreat, Moscow, ID, 3 September.
- 9. Soule, M, LaFrentz S, Oatley M, Krug M, LaFrentz B, Cain KD and Call DR (2004) Comparative genetics of virulent and avirulent strains of Flavobacterium psychrophilum, etiological agent for cold water disease in salmonids. WSU & UI Center for Reproductive Biology, Annual Retreat, ID, June.
- 10. Soule, M (2004) Virulent and avirulent strains of Flavobacterium psychrophilum compared using suppression subtractive hybridization. CVM-VMP-RIP seminar, 20 January.
- 11. Call, DR. 2005. Challenges and opportunities for pathogen detection using DNA microarrays. Critical Reviews in Microbiology 31:91-99.
- 12. LaFrentz, S, N Lindstrom, K Cain, M Soule, DR Call (2005) Development of a sandwich ELISA for detection of Flavobacterium psychrophilum, etiological agent of Bacterial Coldwater Disease in salmonids. WSU Academic Showcase, Pullman, WA, 1 April.
- 27. Cain, K.D., LaFrentz, B.R., Lindstrom, N.M., LaPatra, S.E., and Call, D.R. (2007) Electorphoretic and western blot analyses of the lipopolysaccharide and glycocalyx of Flavobacterium psychrophilum. Flavobacterium 2007 Workshop, May 2-4, Shepherdstown, WV.
- 28. Call, D.R., Soule, M., Shah, D., LaFrentz, S., Cheng, J., Ramsrud, A., Kang, M., LaFrentz, B.R., Cain, K.D., LaPatra, S.E., and Wiens, G.D. (2007) Flavobacterium psychrophilum is composed of two distinct gentetic lineages. Flavobacterium 2007 Workshop, May 2-4, Shepherdstown, WV.
- 29. Chen, J., LaFrentz, S.A., Davis, M.A., LaPatra, S.E., Cain, K., and Call D.R. (2007) Genetic variation of Flavobacterium psychrophilum examined by pulse-field gel electrophoresis. Flavobacterium 2007 Workshop, May 2-4, Shepherdstown, WV.
- 30. Chen Y, Davis MA, LaPatra SE, Cain KD, Snekvik S, and Call DR. (2007). Genetic diversity of Flavobacterium psychrophilum recovered from commercially raised rainbow trout and spawning Coho salmon. 8th Annual College of Veterinary Medicine Research Symposium, Pullman, WA October 17.
- 31. LaFrentz BR, LaPatra SE, Call DR, Cain, KD (2007) Characterization of attenuated strains of Flavobacterium psychrophilum generated by selection for rifampicin resistance. 48th Western Fish Disease Workshop and AFS Fish Health Section Annual Meeting, June 4-6, Jackson Lake Lodge, Grand Teton National Park, WY, USA.
- 32. Lindstrom, N.M., LaFrentz, B.R., Hugunin, H., Call, D.R., and Cain, K.D. (2007) Proteomic analysis of a distinct molecular mass fraction of Flavobacterium psychrophilum. 48th Western Fish Disease Workshop and AFS Fish Health Section Annual Meeting, June 4-6, Jackson Lake Lodge, Grand Teton National Park, WY, USA.
- 33. Lloyd, Snekvik, St-Hilaire, LaPatra, Cain, and Call, 2007. "A Rickettsia-like organism is associated with strawberry disease lesions in rainbow trout." 8th Annual College of Veterinary Medicine Research Symposium, October 17, 2007.
- 34. Lloyd, Hilaire Snekvik, Cain, and Call. 2007. "Identifying the etiological agent of strawberry disease in rainbow trout." WSU Showcase, Pullman, WA, 23 March 2007.
- 35. Lloyd, St-Hilaire, Snekvik, Cain, and Call. 2007. "Identifying the etiologic agent of strawberry disease in rainbow trout." WSU-Wiley Research Symposium, March 2007.
- 36. Plant, K., LaPatra, S., and Cain, K (2007) Heat shock proteins as DNA vaccine candidates against Flavobacterium psychrophilum. 48th Western Fish Disease Workshop and AFS Fish Health Section Annual Meeting, June 4-6, Jackson Lake Lodge, Grand Teton National Park, WY, USA
- 37. Ramsrud, AL, SA LaFrentz, BR LaFrentz, KD Cain, and DR Call. 2007. Differentiating 16S rRNA alleles of Flavobacterium psychrophilum using a simple PCR assay. Journal of Fish Diseases 30:175-180.
- 38. Shah, D.H., Cain, K.D., and Call, D.R. (2007) Effects of codon usage bias on recombinant expression of Flavobacterium psychrophilum proteins in E. coli. Flavobacterium 2007 Workshop, May 2-4, Shepherdstown, WV.
- 13. Soule, LaFrentz, Cain, LaPatra, and Call (2005) Combining suppression subtractive hybridization and microarrays to map the intra-specific phylogeny of Flavobacterium psychrophilum. Western Fish Disease Workshop, Boise, ID, 27-29 June.
- 14. Soule, LaFrentz, Oatley, Krug, LaFrentz, Cain and Call (2005) Comparative genetics of virulent and avirulent strains of Flavobacterium psychrophilum, etiological agent for cold water disease in salmonids. Annual Northwest Reproductive Sciences Symposium, Seattle, WA, 22-23 April.
- 15. Cain, K.D. 2006. Overview of the fish immune system and approaches to vaccine development. Invited seminar. Benchmark Biolabs Incorporated, October 5, 2006.
- 16. Cain KD and Call DR (2006) Comparative genomics and proteomics of Flavobacterium psychrophilum: moving toward vaccine development. Aquaculture ID-WA Research Review, Pullman, WA, March 8-9.
- 17. Call, DR, (2006) Flavobacterium psychrophilum: Etiologic agent of the "other" CWD. Research-in-progress seminar, Department of Veterinary Microbiology and Pathology, WSU, Pullman, WA, September 13.
- 18. Chen, Davis, Cain, and Call. (2006) Genetic variation of Flavobacterium psychrophilum examined by pulse-field gel electrophoresis. 8th Annual College of Veterinary Medicine Research Symposium, October 18.
- 19. LaFrentz BR, Lindstrom NM, LaPatra SE, Call DR, Cain KD (2006) Analysis of Flavobacterium psychrophilum carbohydrate antigens and their potential role in protective immunity. 5th International Symposium on Aquatic Animal Health, September 2-6, San Francisco, CA, USA.
- 20. LaFrentz BR, Lindstrom NM, LaPatra SE, Call DR, Cain KD (2006) An analysis of Flavobacterium psychrophilum carbohydrate antigens and their potential role in protective immunity. 47th Western Fish Disease Workshop, June 26-28, Victoria, BC.
- 21. LaFrentz BR, LaPatra SE, Shewmaker WD, Weighall A, Cain KD (2006) An analysis of Flavobacterium psychrophilum carbohydrate antigens and their potential role in protective immunity. Idaho Chapter of the Wildlife Society, Northwest Section of the Wildlife Society, Northwest Scientific Association, and Northwest Lichenologists Annual Meeting, March 6-8, Boise, ID, USA.
- 22. LaFrentz BR, LaPatra SE, Shewmaker WD, Weighall A, Cain KD (2006) SDS-PAGE and western blot analysis of Flavobacterium psychrophilum carbohydrate antigens and their potential role in protective immunity. Annual Meeting of the Idaho Chapter of the American Fisheries Society, February 15-17, Idaho Falls, ID, USA.
- 23. Maddox, T., Eversman, K., LaPatra, S.E. and Cain, K. D. (2006). Development of autochthonous probiotics to control Flavobacterium psychrophilum, Aeromonas salmonicida, and Yersinia ruckeri in aquaculture, 47th Annual Fish Disease Workshop, Victoria, BC. June 26.
- 24. Cain, K.D. and LaFrentz, B.R. Laboratory Maintenance of Flavobacterium. 2007. Current Protocols in Microbiology. Book chapter - Invited, 6:13B.1.1-13B.1.12.
- 25. Cain KD and Call DR (2007) Comparative genomics and proteomics of Flavobacterium psychrophilum: moving toward vaccine development. Aquaculture ID-WA Research Review, Moscow, ID, January 16-17.
- 26. Cain, K., Sudheesh, P.S., LaFrentz, B.R., Call, D.R., Siems, W.F., LaPatra, S.E., and Wiens, G.D. (2007) Identification of potential vaccine target antigens by immunoproteomic analysis of a virulent and non-virulent strain of the fish pathogen Flavobacterium psychrophilum. Flavobacterium 2007 Workshop, May 2-4, Shepherdstown, WV.
- 39. Wiens, Welch, Rexroad, LaPatra, Call, Hunnicutt, Bhattacharyya, Campbell, and Walunas (2007). Complete genome sequence of Flavobacterium psychrophilum strain CSF 259-93 and characterization of a large cluster of genes encoding leucine-rich repeats. Flavobacterium 2007 Workshop, May 2-4, Shepherdstown, WV.
- 40. Overturf, K., LaPatra, S., Towner, R., Campbell, N., Narum, S. 2010. Relationships between growth and disease resistance in rainbow trout (Oncorhynchus mykiss). Journal of Fish Diseases 33:321-329.
- 41. Purcell, M.K., K.J. Laing, J. Woodson, G. Thorgaard, and J.D. Hansen. 2009. Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes. Fish and Shellfish Immunology. 26: 293-304.
- 42. Lloyd, SJ, SE LaPatra, KR Snekvik, KD Cain, and DR Call. 2011. Quantitative PCR demonstrates a positive correlation between a Rickettsia-like organism and severity of strawberry disease lesions in rainbow trout (Oncorhynchus mykiss). Journal of Fish Diseases 34:701-709.
- 43. Call, DR, KR Snekvik, and KD Cain. Strawberry disease Research. Waterlines, Spring 2010, p9. Lloyd, SJ, KR Snekvik, S St-Hilaire, SE LaPatra, KD
- 44. Cain, and DR Call. 2008. Strawberry disease lesions in rainbow trout (Oncorhynchus mykiss) are closely associated with a Rickettsia-like organism. Disease of Aquatic Organisms 82:111-118. PMID: 19149374.
- 45. Metselaar, M, KD Thompson, RML Gratacap, MJL Kik, SE LaPatra, SJ Lloyd, DR Call, PD Smith, and A Adams. 2010. Association of red-mark syndrome with a Rickettsia-like organism and its connection with strawberry disease in the USA. Journal of Fish Diseases 33:849-858. PMID: 20854353.
- 46. Barroso, R.M., Wheeler, P.M., LaPatra, S.E., Drew, R.E. & Thorgaard, G.H. 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.
- 51. Call, DR (2004) Application of DNA microarrays to pathogen detection and comparative genomics. Fifth Annual General Meeting of the Genome and Health Initiative, Montreal, Canada, 16-19 May.
- 52. Call DR and Cain KD (2004) Comparative genomics and proteomics for identification of virulence and immunogenic antigens. Aquaculture, ID-WA Research Review, Moscow, ID, 4 Feb 2004.
- 53. 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.
- 54. LaFrentz, B.R., LaPatra, S.E., Call, D.R., Wiens, G.D., and Cain, K.D. 2011. Identification of Immunogenic proteins within distinct molecular mass fractions of Flavobacterium psychrophilum. Journal of Fish Diseases (In Press)
- 47. Campbell, N., Overturf, K. and Narum, S. 2009. Characterization of 22 novel single nucleotide polymorphism markers in steelhead and rainbow trout. Molecular Ecology Resources 9:318-322.
- 48. Call, DR, MK Borucki and FJ Loge. 2003. Detection of bacterial pathogens in environmental samples using DNA microarrays. Journal of Microbiological Methods 53:235-243.
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Progress 08/15/08 to 08/14/09
Outputs
OUTPUTS: 1. Flavobacterium psychrophilum: We tested vaccines to prepare protein for immunization. GldN, which is involved in gliding motility of F. psychrophilum, showed early promise as an antigen, but there was no evidence of protection. In another trial we combined seven proteins as a single formalin killed bacterin. Again, no evidence of protection. We also constructed a fusion protein, but could not produce enough for an experiment. 2. Autochthonous probiotics: We have found 10 probiotic isolates shown to inhibit growth of F. psychrophilum. Two of have shown a decrease in mortality and have been identified as strains of Enterobacter species. Field trials have shown a 40-50% reduction in mortality. 3. Strawberry Disease in rainbow trout: We screened SD lesions and identified a DNA sequence from a Rickettsia-like organism (RLO). Testing showed that it was associated with SD lesions. We developed a nested-PCR assay for the RLO marker and developed a quantitative PCR assay. Using this, we demonstrated that RLO sequence copy number is correlated with lesion severity. We also tested the possibility that a condition called red-mark syndrome (RMS) was associated with the RLO marker and demonstrated that RMS lesions are positive for the RLO. A study was begun to compare the lesions from SD with two related conditions (RMS and warm-water SD). This work represents the first systematic effort to characterize the lesions from this group of conditions. 4.Mitochondrial and Maternal Effects on Growth in Rainbow Trout: We produced Hot Creek clonal trout from egg sources with different mitochondrial haplotypes. No significant differences were detected in length, weight, or condition factors among the three haplotypes. 5. Sex Determination in White Sturgeon: Efforts focused on identification of sex markers for white sturgeon from males and females from a single-pair mating (05D). A total of 13 primer combinations gave results suggesting female linkage of sex determination and none gave results suggestive of male linkage. When these results were tested, three types of results were found. 1. Several of the combinations did not show sex associations. 2. A few combinations showed the female-specific bands (3 to 6 individuals) among the total of 20 females and 20 males screened. 3. A few combinations revealed a quantitative difference in band frequency between females and males. 6. Genetic Typing and Mapping of the Rainbow Trout Genome: This project is using DNA sequencing to identify single nucleotide polymorphisms and to characterize genome structure in rainbow trout (Oncorhynchus mykiss). A rainbow trout "SNP chip" is currently being developed. Work has proceeded on identification of SNPs from transcribed genes in eyed embryos and head kidney. We are looking for a better way to gain a higher success rate at predicting single copy genes. Several immune genes and SNPs have been identified. Work has proceeded on sequencing bacterial artificial chromosomes (BACs) containing metallothionein gene duplicates with a known gene phylogeny. This study used de novo genomic sequence assembly on 14 BACs representing the four members of the estrogen receptor family. PARTICIPANTS: Washington State University: Douglas Call, Jim Derringer, Amit Dhingra, Karol Gliniewicz, Amy Long, Ruth Phillips, Devendra Shah, Kevin Snekvik, Gary Thorgaard University of Idaho: Ken Cain, David Burbank, Kurt Eversman, Tarah Johnson, Nicole Lindstrom, Timberly Maddox, James Nagler, Karen Plant, Barrie Robison. Other Personnel: Gary Fornshell, UI Extension; Scott LaPatra, Clear Springs Foods. TARGET AUDIENCES: Target audiences are primarily the industries that are involved in freshwater trout, sturgeon, and marine estuary shellfish cultivation. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1. Flavobacterium psychrophilum: FP1493 was identified as a potential vaccine. In the first trial too few fish died in the positive control tank. A second trial is in progress. We have two proteins ready for testing and have constructed the whole-genome expression library for F. psychrophilum. We will transfer the library to our Vibrio parahaemolyticus expression host, induce expression en mass, and harvest protein for testing. 2. Autochthonous probiotics: Initial experiments using C6-6 showed that fish exhibited no mortality following challenge with F. psychrophilum and resulted in a reduction in mortality due to CWD. A second probiotic (C6-8) also demonstrated a reduction in mortality. In three separate hatchery trials, rainbow trout fry were fed our probiotic and mortality was reduced by nearly 50% following a natural outbreak of CWD. Such results demonstrate the commercial potential. 3: Strawberry Disease in trout: Fluke eggs were observed in the gills of SD-affected fish. This led to the proposition that the RLO is linked to the fluke infestation. It has been postulated that we should be able to identify the RLO marker in snails from affected raceways. We confirmed this prediction. This observation also suggests that we culture the RLO and this work is underway. With additional funding, we will co-house pathogen free trout with infected snails to determine if we can induce SD. We will also attempt to isolate the RLO from flukes that are isolated from infected snails and propagate RLO in the lab. If the hypothesis is confirmed, SD control will be linked to control of snails. 4. Mitochondrial and Maternal Effects on Growth in Trout: We identified differences in condition factor between our clonal lines. One line has a higher condition factor (heavier weight for the same length) than does the other. We are analyzing crosses to determine whether major loci are associated with the condition factor difference. We also developed a species identification test for use with degraded DNA. These are likely to be useful for evolutionary studies involving ancient DNA samples. 5. Sex Determination in Sturgeon: Results are focusing on analysis of markers in individuals. This has revealed the sturgeon genome to apparently contain a lower proportion of markers behaving as disomic (diploid) Mendelian segregating loci. Many markers have been found in only a low proportion of the siblings, presumably reflecting a polyploid inheritance pattern. These low-ratio segregating markers will be incorporated during mapping to characterize inheritance. Our initial efforts suggest a female heterogametic mechanism in this species. However, the mechanism also appears to be genetically complex and may involve multiple chromosome regions. 6. Typing and Mapping of the Trout Genome: The addition of whole genome 5kb mate-paired sequence, ER cDNA and BES sequence allowed orientation of contigs for individual ER BACs. The Rainbow Trout Physical map allowed us to identify the additional 12 BACs needed to complete the ERb sequencing. Libraries for these additional 12 BACs are being prepared for Illumina sequencing to complement the 454 dataset. Data coming soon.
Publications
- Burbank, D. and Cain, K.D. (2008). Assessing Candidate Probiotic Use for the Control and Prevention of Flavobacterium psychrophilum in Rainbow Trout (Oncorhynchus mykiss). McNair Research symposium, University of Idaho, July 31.
- Cain, KD. (2008) Coldwater Disease Research Update: Vaccine Development. Idaho Aquaculture Association Annual Meeting, June 21, Twin Falls, ID
- Cain, KD, and LaFrentz, BR (2008) Defining acquired immunity to Flavobacterium psychrophilum: implications for developing a coldwater disease (CWD) vaccine. 8th International Congress on the Biology of Fish, July 28-August 1, Portland, OR, USA
- Lloyd, SJ, KR Snekvik, S St-Hilaire, SE LaPatra, KD Cain, and DR Call. 2008. Strawberry disease lesions in rainbow trout (Oncorhynchus mykiss) are closely associated with a Rickettsia-like organism. Disease of Aquatic Organisms 82:111-118. PMID: 19149374.
- Lloyd SJ, LaPatra SE, Snekvik KR, Lindstrom N, St-Hilaire S, Cain KD, Call DR. 2008. A Rickettsia-like organism is associated with strawberry disease lesions in rainbow trout. Department of Veterinary Microbiology and Pathology Research in Progress Seminar, College of Veterinary Medicine, Washington State University, Pullman WA. Oral presentation.
- Lloyd, LaPatra, Snekvik, Lindstrom, Cain, and Call. 2008. Possible isolation in tick cell culture of a Rickettsia-like organism associated with Strawberry Disease lesions in rainbow trout. 9th Annual College of Veterinary Medicine Research Symposium, October 18, 2008. Posters.
- Lloyd, LaPatra, Snekvik, St-Hilaire, Cain, and Call. 2008. Strawberry disease lesions in rainbow trout are associated with a Rickettsia-like organism. Oral presentation. American Fisheries Society Fish Health Section Annual Meeting, Charlottetown, Prince Edward Island, June 2008.
- Lloyd, LaPatra, Snekvik, St-Hilaire, Cain, and Call. 2008. Strawberry disease lesions in rainbow trout are associated with a Rickettsia-like organism. Oral Presentation. 49th Western Fish Disease Workshop, Ocean Shores, WA, June 23-25, 2008.
- Lloyd, LaPatra, Snekvik, St-Hilaire, Cain, and Call. 2008. Strawberry disease lesions in rainbow trout are associated with a Rickettsia-like organism. Oral Presentation. American Fisheries Society Washington - British Columbia Chapter (formerly North Pacific International Chapter) Annual General Meeting, Bellingham WA, 2008.
- Lloyd, Snekvik, LaPatra, St-Hilaire, Cain, and Call. 2008. A Rickettsia-like bacterium is associated with strawberry disease lesions in rainbow trout. WSU Showcase, Pullman, WA, 28 March 2008.
- Shah, DH, Cain KD, Wiens G, and Call DR (2008). An alternate expression system for the production of recombinant Flavobacterium psychrophilum proteins for development of a cold water disease vaccine in rainbow trout. American Fisheries Society Fish Health Section Annual Meeting, Charlottetown, Prince Edward Island, June.
- Wiens, G, LaPatra SE, Call DR, Welch T. (2009). Comparative genomics of the Flavobacterium psychrophilum frp locus. American Society for Microbiology, General Meeting, Philadelphia, PA, May 18-22.
- Gliniewicz, K, Cain KD, Snekvik K, and Call DR (2009). The role of rpoB in the attenuation of Flavobacterium psychrophilum after passage with rifampicin. 10th Annual College of Veterinary Medicine Research Symposium, Pullman, WA, October 14.
- Call, DR (2009) Detection and differentiation of bacterial pathogens using nucleic acid based assays. Invited seminar, University of Minnesota, December 3.
- Lloyd, S, S LaPatra, K Snekvik, N Lindstrom, K Cain, and DR Call. 2009. Possible isolation in tick cell culture of a Rickettsia-like organism associated with Strawberry Disease lesions in rainbow trout. WSU Showcase, Pullman, WA, 27 March 2009.
- Metselaar, Thompson, Kik, LaPatra, Lloyd, Call, Adams. 2009. "Studies on red mark syndrome." European Workshop on PhD and Post-doctoral Fellows on Anadromous Salmonids (NoWPaS), March 19-22, 2009, Southampton, England.
- Burbank, D.R., LaPatra, S.E., Fornshell, G. and Cain, K.D. 2010. Isolation of bacterial probiotic candidates from the gastrointestinal (GI) tract of rainbow trout and screening for in vitro inhibitory activity to Flavobacterium psychrophilum. Journal of Fish Diseases (Submitted)
- Cain, K.D., Burbank, D.R., Wilson, C. and LaPatra, S.E. (2010). Assessing candidate probiotic use for the possible control of Flavobacterium psychrophilum in rainbow trout. Idaho Aquaculture Association annual meeting, Twin Falls, ID June 12th.
- Call, DR, KR Snekvik, and KD Cain. 2010. Strawberry disease Research. Waterlines, Spring 2010, p9.
- Gliniewicz, Snekvik, Cain, LaPatra and Call, 2010. Assessing the immune-protective potential of FP1493 against coldwater disease in rainbow trout Washington State University Showcase, March 2010.
- Gliniewicz, Snekvik, Cain, LaPatra, and Call, 2010. Assessing the immune-protective potential of FP1493 against coldwater disease in rainbow trout. American Society for Microbiology, San Diego, CA, 23-27 May 2010.
- Gliniewicz, Cain, Snekvik, and Call. 2010. The role of rpoB gene in rifampicin attenuation of Flavobacterium psychrophilum. 11th Annual College of Veterinary Medicine Research Symposium, October 27, 2010.
- Johnson, T.J., Parvez, N., Call, D.R., LaFrentz, B.R., Miura, T., and Cain, K.D. 2010. Confirmation of Safety and Efficacy for a Live Attenuated Flavobacterium psychrophilumVaccine. Sixth Intl. Symposium of Aquatic Animal Health, September 5-9th, Tampa, FL.
- Johnson, TJ, BR. LaFrentz, DR. Call, and KD Cain. 2010. Characterization of an attenuated Flavobacterium psychrophilum vaccine. Idaho Chapter of the American Fisheries Society Annual Meeting, Pocatello, ID March 2-5.
- Jordan, L.G., C.A. Steele and G.H. Thorgaard, 2010. Universal mtDNA primers for species identification of degraded bony fish samples. Molecular Ecology Resources 10: 225-228.
- Lanier, Shah, Kumar, LaPatra, Gliniewicz, Snekvik, Cain, and Call, 2010. Production of recombinant in vivo induced proteins of Flavobacterium psychrophilum for development of a coldwater disease vaccine in rainbow trout. Washington State University Showcase, March 2010.
- Lloyd, SJ, SE LaPatra, KR Snekvik, KD Cain, and DR Call. 2010. Quantitative PCR demonstrates a positive correlation between a Rickettsia-like organism and severity of strawberry disease lesions in rainbow trout (Oncorhynchus mykiss). Diseases of Aquatic Organisms (in review)
- Metselaar, M, KD Thompson, RML Gratacap, MJL Kik, SE LaPatra, SJ Lloyd, DR Call, PD Smith, and A Adams. 2010. Association of red-mark syndrome with a Rickettsia-like organism and its connection with strawberry disease in the USA. Journal of Fish Diseases 33:849-858. PMID: 20854353.
- Plant, K.P., LaPatra, S.E., Call, D.R. and Cain, K.D. 2010. Is vaccination with Flavobacterium psychrophilum gliding motility protein N (GldN) effective American Fisheries Society (Fish Health Section) 51st Annual Western Fish Disease Workshop, Corvallis, OR June 22-24.
- Plant, K.P., LaPatra, S.E., Call, D.R., and Cain, K.D. 2010. Immunization of rainbow trout (Oncorhynchus mykiss) with Flavobacterium psychrophilum proteins elongation factor-Tu, SufB Fe-S assembly protein and ATP synthaseb. Journal of Fish Diseases (In Press)
- Polinski, M.P., Fehringer, T.R., Johnson, K.A., Snekvik, K.R., LaPatra, S.E., LaFrentz, B.R., Ireland, S.C., Cain, K.D. 2010. Characterization of susceptibility and carrier status of burbot to IHNV, IPNV, Flavobacterium psychrophilum, Aeromonas salmonicida, and Renibacterium salmoninarum. Journal of Fish diseases 33, 559-570.
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