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    WRAC ANNUAL PROGRESS REPORT – Year 3 PART I: Summary PROJECT TITLE: Coldwater disease prevention and control through vaccine development and diagnostic improvements REPORT GIVEN IN YEAR: 2010 REPORTING PERIOD: September 1, 2009 – August 31, 2010 (3rd year of project) AUTHOR: Ken Cain and Doug Call FUNDING LEVEL: First yr funding: $81,555 (received February 2008) Second yr funding: $80,043 (received March 2009) Third yr funding: $81,637 (received April 2010) Fourth yr funding: $81,639 (approved 10/09) PARTICIPANTS: Kenneth Cain* (Work Group Chair) University of Idaho Douglas Call* Washington State University Scott LaPatra Clear Springs Foods, Inc. Gary Fornshell* University of Idaho Greg Weins USDA, West Virginia Technical Advisor: Gael Kurath USGS, Washington Industry Advisor: Jim Parsons Troutlodge, Washington Graduate Students: Amy Long/Karol Gliniewicz UI/WSU Faculty participant: Devendra Shah Washington State University PROJECT OBJECTIVES: The goals of this project are to evaluate strategies that would aid in developing more effective ways of managing coldwater disease (CWD) at aquaculture facilities, and to identify possible bacterial genes that may be targeted for vaccine development and testing. Presently, disease management is difficult at many facilities and there is no commercial vaccine available for Flavobacterium psychrophilum, the causative agent for CWD. The specific objectives for this project are: 1. Identify potential vaccine candidates using comparative proteomic analysis of an attenuated strain of Flavobacterium psychrophilum and determine if crude cell lysate can be used as a subunit vaccine delivery vehicle in the absence of adjuvant. • Candidate recombinant proteins will be tested in vaccine trials. 2. Validate quantitative diagnostic assays (ELISA and ovarian fluid filtration FAT). • Correlate assay results to risk of vertical transmission or disease susceptibility • Establish threshold levels for culling broodstock and/or eggs 3. Based on results from objective 2:


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    •Develop other assays (e.g. real-time quantitative PCR) for quantification of infection in ovarian fluid. 4. Develop an integrated outreach program to meet stakeholder needs. • Based on results obtained from this project and the number of deliverables made available to researchers and/or the aquaculture community, a number of outreach/extension products will be developed related to prevention and/or control of CWD through vaccination or implementation of new disease management strategies at broodstock facilities. ANTICIPATED BENEFITS: Coldwater disease (CWD) has become one of the most significant disease problems in commercial trout aquaculture in recent years. It is a worldwide problem, and in the Pacific Northwest alone, losses from CWD can range from 18% to 30% with estimated economic impacts in Idaho alone reaching approximately $10 million. In addition to the trout industry, federal, state, and tribal hatcheries rearing a variety of salmonids (steelhead and Coho salmon in particular) also suffer dramatic losses. The ability to manage around the disease by culling eggs from heavily infected broodstock would likely provide an overall reduction of disease incidence at a facility. This may result from limiting the pathogen’s ability to be vertically transmitted to progeny through the egg, or from eliminating broodstock carriers and providing an overall reduction of pathogen presence at facilities. This approach has worked well for bacterial kidney disease (BKD). In addition to benefits associated with developing improved disease management strategies, identifying antigens that may be targeted for vaccine development will be important. If effective vaccine targets are identified then the long-term goal of developing a commercial CWD vaccine would provide a tool to prevent CWD at aquaculture facilities. Currently, such preventative measures do not exist and control relies on antibiotic use. Anticipated benefits associated with this project will include the availability of additional diagnostic tools (monoclonal antibodies and pathogen detection assays) for broodstock and/or egg culling to minimize CWD outbreaks, identification of potential vaccine candidates, and subsequent reduction of mortalities due to CWD. PROGRESS AND PRINCIPAL ACCOMPLISHMENTS: Funding for this project became available in February 2008, and a PhD student (Amy Long) was recruited in May 2008, a Postdoctoral Fellow (Rajesh Kumar) worked on this project from July 2008 to July 2009, and a PhD student (Karol Gliniewicz) has recently joined Dr. Call’s lab (2009). A program review of the project was conducted by Dr. Jerri Bartholomew in May 2010 and progress was reported to the WRAC board. The primary workgroup members were involved in this review and it offered an opportunity to confirm upcoming plans and discus results to date. A more formal workgroup meeting will be scheduled in November following the next round of sample collections at Troutlodge (currently scheduled for mid-October). Objective 1: Identify potential vaccine candidates using comparative proteomic analysis of an attenuated strain of Flavobacterium psychrophilum and determine if crude cell lysate can be used as a subunit vaccine delivery vehicle in the absence of adjuvant.


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    The original project proposal included efforts to develop and screen an IVIAT library to detect in vivo expressed antigens. As reported last year, while we were able to detect in vivo expressed proteins using large culture volumes, we were unable to obtain sufficient analytic sensitivity to differentiate in vivo expressed proteins from background when using a high-throughput format. Lacking this higher throughput, there was no practical means to screen the library. We received approval to change our focus for this component of the project to employ comparative proteomics to identify other candidate proteins based on differential expression from a virulent strain (CSF259.93) and an attenuated strain (CSF259.93.B17; “B17”). We also proposed to determine if subunit vaccine candidates can be screened using crude lysate from our Vibrio parahaemolyticus expression host. The intent of this second component was to improve the efficiency of subunit testing and reduce overall cost of vaccine administration. Finally, time and resources permitting, we proposed to initiate work to determine why B17 is attenuated with the expectation that this would lead to a more rapid means to develop repeatable and stable attenuation in F. psychrophilum and other bacterial pathogens. Two-dimensional gel electrophoresis experiments demonstrated that a number of proteins are differentially expressed when B17 and CSF259.93 are compared. We have now identified 12 proteins of interest and are in the process of expressing recombinant proteins and verifying their antigenicity. Based on earlier work, we rely heavily on a strain of Vibrio parahaemolyticus (strain NY-4) as our host bacterium for expression of recombinant F. psychrophilum proteins. V. parahaemolyticus usually works better than E. coli and we hypothesize that this is due to a closer match between codon usage for F. psychrophilum and V. parahaemolyticus compared with E. coli. Listonella angullarum, which is closely related to Vibrios, has been used successfully as bacterin without addition of adjuvant. Consequently, we hypothesized that we could used crude lysate from V. parahaemolyticus as the vehicle for delivering recombinant protein in lieu of relatively expensive purification procedures. Our first two trial experiments indicated some support for this hypothesis, but our most recent trial indicates that delivery of recombinant protein in Vibrio lysate by itself is insufficient. Not only did we find no protective response, but a partial analysis of antisera from convalescent fish indicated that lysate inoculated fish produced a minimal antibody response. Consequently, we have rejected our hypothesis and will continue screening subunit vaccine candidates in conjunction with FCA. Our efforts for Objective 1 will focus on completing work to identify and test immunologically reactive antigens for vaccine development. Furthermore, using data from the differentially expressed proteins we can now proceed with efforts to determine the mechanism by which B17 is attenuated. Objective 2: Validate quantitative diagnostic assays (ELISA and ovarian fluid (membrane) filtration FAT). Optimization of the capture ELISA was completed this past year. A challenge experiment was conducted to relate infection levels to disease in fish infected with Flavobacterium psychrophilum. Optical density (O.D) values for positive samples ranged from 0.102 to 0.209 (7.86 x 104 – 2.16 x 105 CFU ml-1) throughout the challenge. Interestingly, no mortality or gross clinical signs of BCWD were observed, confirming that our assay is capable of detecting subclinical F. psychrophilum infections.


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    In February 2010, ovarian fluid and tissue samples were collected from 60 female broodstock at Troutlodge. Samples were tested for F. psychrophilum using nested PCR (nPCR), membrane filtration fluorescent antibody test (MF-FAT), culture, and capture ELISA. The results of the assays were used to select eggs from five families to use in the proposed experiments. All broodstock were infected, but infection levels appeared low when all assays were evaluated together. We selected one family positive only by MF-FAT and four families that were positive by at least two of our assays. Eyed eggs from each family were sent to the University of Idaho and reared in our wetlab facility. Progeny were sampled on a weekly basis and tested by nPCR for F. psychrophilum. Disinfected eyed eggs and fry tested positive for F. psychrophilum by nPCR. However, YPB growth on TYES-TB plates was not observed until Day 36. Once fry reached 0.5 g, controlled stress experiments were initiated in an attempt to induce a BCWD outbreak. Two different stressors were used: chronic gas supersaturation and handling stress. Mortalities occurred in all families but there was no significant difference between families. Random sampling of all tanks was conducted on a weekly basis and tissue samples used for nPCR. While a confirmed outbreak of BCWD did not occur, we did show an increase in the proportion of fish that tested positive for F. psychrophilum in each family. This suggests that fish had not cleared the infection prior to initiating the stress experiment and that F. psychrophilum infection once again increased to detectable levels. Samples were also collected from regional hatcheries this year. Steelhead returning to Wallowa Hatchery (WH) (Enterprise, OR) were sampled in Spring 2010. Sixty fish were sampled over six weeks, 10 fish per sampling date. Twenty-three percent of kidney samples were positive by the capture ELISA with the estimated CFU ml-1 ranging from 4.92 x 104 to 2.34 x 106. Samples collected from coho salmon spawned in Fall 2003 at two different hatcheries in Washington State were also received to compare against previous ELISA results reported by Lindstrom et al. (2009). Prevalence of infection at the two facilities was 35% and 100%, and estimated CFU ml-1 of these samples ranged from 3.85 x 104 to 9.09 x 105. Objective 3: Based on results from objective 2: Develop other assays (e.g. real-time quantitative PCR) for quantification of infection in ovarian fluid. The gene encoding the antigen on the outer membrane of F.psychrophilum that is recognized by MAb FL43 was sequenced by the Call lab this year. Once the nucleotide sequence was known, primers for a Sybr Green assay were developed. Using a TOPO® TA cloning kit, a section of FP1493 was cloned onto a plasmid. The plasmid copy of the gene was then used to optimize qPCR conditions and determine primer efficiency. Objective 4: Develop an integrated outreach program to meet stakeholder needs. Outreach activities have resulted in two articles in Waterlines describing this WRAC project. Project deliverables resulting from this work so far include the commercialization of monoclonal antibody FL43 through Immunoprecise Antibodies, Inc. Labeled antibodies are now being sold to researchers and aquaculture facilities interested in improved diagnostics for BCWD. Protocols for ELISA and FAT assays developed in our lab were provided to Immunoprecise and are included when antibody is sold.


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    USEFULNESS OF FINDINGS: Progress in diagnostic improvements is moving forward very well and should result in correlation of assays to disease risk in progeny from infected broodstock. Such finding would increase the need for labs to purchase the commercialized antibody and utilize assay protocols. Vaccine work has shifted from original objectives, but as we begin to define attenuation mechanisms this could lead to more efficient ways to create vaccines strains for F. psychrophilum and possibly other fish bacterial pathogens. WORK PLANNED FOR NEXT YEAR: Objective 1: Identify and test potential vaccine candidates based on comparative proteomic analysis of CSF259.93 and CSF259.93.B17 and determine the mechanism of B17 attenuation. We have completed the proteomic analysis and with the exception of some material currently in process, we do not plan to identify any additional differentially expressed proteins. At this time we will proceed with expression of recombinant proteins and verify antigenicity by western blot using antisera from convalescent rainbow trout. Antigenic proteins will enter our testing “pipeline” that involves immunizing trout followed by challenge with strain CSF259.93 (e.g., Sudheesh et al. 2007). Importantly, each immunization trial requires approximately three months to execute and there are usually only sufficient fish and tanks available to complete one immunization trial at a time. Thus, in the time remaining in this project we will not be able to assess every protein identified in this study. Instead, we will focus on proteins that are immunologically reactive and that are probable virulence factors such as the Fpp2 (Table 1 see detail section) although this will depend in part on our ability to produce a sufficient mass of the protein of interest. The expected deliverable for this segment of our project is a manuscript describing the differentially expressed proteins and, if successful, a manuscript describing identification of a vaccine candidate that induces significant protective immunity. We will also proceed with efforts to determine why strain B17 is attenuated. Our working hypothesis is that attenuation results from altered global transcriptional regulation as evidenced by a significant change in protein expression pattern relative to the pathogenic parental strain CSF259-93. Altered expression is probably a result of a point mutation in the beta subunit (rpoB) of the RNA polymerase, and this year we have confirmed that one of the expected mutations is present in B17. We predict that the pattern of altered protein expression is repeatable, predictive of attenuation, and can be engineered independently from rifampicin passage. The mechanism underlying altered protein expression most likely involves a change in how the RNA polymerase binds promoter sequences or a change in how the RNA polymerase interacts with key transcriptional factors (sigma factors). There is insufficient time (1 year) and resources remaining for this project to fully test both mechanisms. Nevertheless, we are well positioned to determine if promoter sequence binding plays a role in this process. That is, we will determine if mutations in the rpoB affect the efficiency of promoter binding using the differentially regulated genes as “bait” for this assay. This will be followed by introducing an identical mutation into a pathogenic strain of F. psychrophilum to recapitulate attenuation. The rationale for this series of experiments is that we will identify the specific mechanism involved in rifampicin attenuation (currently a knowledge gap), which will provide the opportunity to generate a genetically engineered and potent live-attenuated strain that will also be unlikely to revert to virulence.


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    Knowledge of the precise mechanism of rifampicin attenuation may also offer a possibility of creating of attenuated strains of other bacterial pathogens Objective 2: Validate quantitative diagnostic assays (ELISA and ovarian fluid (membrane) filtration FAT). Validation of the diagnostic assays will continue this year. We anticipate sampling again at Troutlodge this fall and screening 60 female broodstock for F. psychrophilum using the standard diagnostic assays. As before, five families will be selected for further experiments. In addition, we will continue to collect samples from regional hatcheries. Samples will be collected in Fall 2010 from Skookum Creek Fish Hatchery (Acme, WA), a coho salmon spawning facility, and in Spring 2011 from WH. Not only do the samples from naturally returning fish aid in validating our assays but we can also use the data to compare the prevalence of F. psychrophilum in coho salmon, rainbow trout, and steelhead. Objective 3: Based on results from objective 2: Develop other assays (e.g. real-time quantitative PCR) for quantification of infection in ovarian fluid. Optimization of the qPCR assay will continue in the upcoming year. Sensitivity and specificity of this qPCR assay will be evaluated. Once that has been done, we will focus on DNA extraction from ovarian fluid and eliminate any possible inhibition. Ovarian fluid samples that have been collected from various hatcheries over the past 2 years have been stored at -80°C and will used in the qPCR assay. This will allow us to evaluate the efficiency of the assay as well as the compare results to those obtained by other assays for the same samples. In addition to the above assays, we have recently partnered with Infoscitex, a biotech company, who was awarded a Phase I USDA/SBIR grant to develop new diagnostic assays for F. psychrophilum that use aptamers as the detection molecules. Aptamers are short sequences of DNA or RNA nucleotides that can be used in place of classical antibodies in ELISA assays. Reporter molecules can also be added to aptamers so that they can be used in qPCR. Phase 1, production of an aptamer specific to FP1493 (the protein recognized by FL43) is underway. Such assays have the potential for greater sensitivity than those currently being tested. Objective 4: Develop an integrated outreach program to meet stakeholder needs. Based on project deliverables, evaluate need for workshops and develop a draft WRAC Extension publication. Continue to work with Immunoprecise to inform stakeholders of the availability of improved diagnostic tools. IMPACTS: The primary impact is the commercialization of monoclonal antibody FL43 through Immunoprecise Antibodies, Inc. This is now being sold to research labs and/or aquaculture companies in the un-conjugated form or conjugated to FITC or HRP. Protocols for the capture ELISA and FAT have been distributed to fish health labs in the region. Furthermore, we have provided these protocols to ImmunoPrecise to be distributed to customers when they purchase FL43.


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    SUPPORT: WRAC-USDA Other Total Year University Industry Other Total Funding Federal Support 2008 $81,555.00 $81,555.00 2009 $80,043.00 $80,043.00 2010 $81,637.00 $81,637.00 $81,639 $81,639 2011 (approved 10/09) Total $324,874.00 PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: Refereed publications: LaFrentz, BR, SE LaPatra, DR Call, GD Wiens, and KD Cain. 2009. Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media. Diseases of Aquatic Organisms 87:171-182. PMID: 20099411. 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 21:43-56. PMID: 19485125. 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): p. 521-34 Plant, KP, SE LaPatra, DR Call, and KD Cain. In review. Immunization of rainbow trout (Oncorhynchus mykiss) with Flavobacterium psychrophilum proteins elongation factor-Tu, SufB Fe-S assembly protein and ATP synthaseb. General articles: Cain, K.D. 2009. Strategies for Control and Prevention of Coldwater Disease. Waterlines newsletter 15 (1): p. 18-20 Cain, K.D. Call, D.R, and Snekvik, K.R. 2010. A tail of two diseases (Coldwater disease and Strawberry disease research) Waterlines newsletter 16 (1): p. 10-11 Presentations: Gliniewicz, Cain, Snekvik, and Call, “The role of rpoB in the attenuation of Flavobacterium psychrophilum after passage with rifampicin” Poster presented at the 10th Annual College of Veterinary Medicine Research Symposium, October 14, 2009.


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    Long, A., Call, D.R., and Cain, K.D. 2009. Comparison of diagnostic techniques for detection of Flavobacterium psychrophilum in ovarian fluid. Talk presented at the 50th Western Fish Disease Workshop and AFS Fish Health Section Annual Meeting. Park City, Utah. June 7-10. Gliniewicz, K, K Snekvik, K Cain, S LaPatra, and D Call. Assessing the immune-protective potential of FP1493 against coldwater disease in rainbow trout. Poster presented at American Society for Microbiology general meeting, May 2010, San Diego, CA. Lanier, A, R Kumar, S LaPatra, K Gliniewicz, K Snekvik, K Cain, D Shah, and D Call. Production of recombinant in vivo induced proteins of Flavobacterium psychrophilum for development of a cold water disease vaccine for rainbow trout. Poster presented at the WSU Showcase, March 2010, Pullman, WA. Long, A., Call, D.R., and Cain, K.D. 2010. Use of Diagnostic Assays to Screen Rainbow Trout (Oncorhynchus mykiss) Broodstock for Flavobacterium psychrophilum. Talk presented at the 6th International Symposium for Aquatic Animal Health and AFS Fish Health Section Annual Meeting. Tampa, Florida. September 5-9. SUBMITTED BY: ____________________________________September 8, 2010____ Title: (Work Group Chair or PI) Date September 13, 2010 APPROVED: _____________________________________________________ Technical Advisor (if Chair’s report) Date


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    WRAC ANNUAL PROGRESS REPORT – Year 3 PART II: Detail PROJECT TITLE: Coldwater disease prevention and control through vaccine development and diagnostic improvements REPORT GIVEN IN YEAR: 2010 REPORTING PERIOD: September 1, 2009 – August 31, 2010 (3rd year of project) AUTHOR: Ken Cain and Doug Call FUNDING LEVEL: First yr funding: $81,555 (received February 2008) Second yr funding: $80,043 (received March 2009) Third yr funding: $81,637 (received April 2010) Fourth yr funding: $81,639 (approved 10/09) PARTICIPANTS: Kenneth Cain* (Work Group Chair) University of Idaho Douglas Call* Washington State University Scott LaPatra Clear Springs Foods, Inc. Gary Fornshell* University of Idaho Greg Weins USDA, West Virginia Technical Advisor: Gael Kurath USGS, Washington Industry Advisor: Jim Parsons Troutlodge, Washington Graduate Students: Amy Long/Karol Gliniewicz UI/WSU Faculty participant: Devendra Shah Washington State University PROJECT OBJECTIVES: 1. Identify potential vaccine candidates using comparative proteomic analysis of an attenuated strain of Flavobacterium psychrophilum and determine if crude cell lysate can be used as a subunit vaccine delivery vehicle in the absence of adjuvant. a. Candidate recombinant proteins will be tested in vaccine trials. 2. Validate quantitative diagnostic assays (ELISA and ovarian fluid filtration FAT). • Correlate assay results to risk of vertical transmission or disease susceptibility • Establish threshold levels for culling broodstock and/or eggs 3. Based on results from objective 2: • Develop other assays (e.g. real-time quantitative PCR) for quantification of infection in ovarian fluid. 4. Develop an integrated outreach program to meet stakeholder needs. • Based on results obtained from this project and the number of deliverables made available to researchers and/or the aquaculture community, a number of


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    outreach/extension products will be developed related to prevention of CWD and tailoring disease management at broodstock facilities. ANTICIPATED BENEFITS: Coldwater disease (CWD) has become one of the most significant disease problems in commercial trout aquaculture in recent years. It is a worldwide problem, and in the Pacific Northwest alone, losses from CWD can range from 18% to 30% with estimated economic impacts in Idaho alone reaching approximately $10 million. In addition to the trout industry, federal, state, and tribal hatcheries rearing a variety of salmonids (steelhead and Coho salmon in particular) also suffer dramatic losses. The ability to manage around the disease by culling eggs from heavily infected broodstock would likely provide an overall reduction of disease incidence at a facility. This may result from limiting the pathogen’s ability to be vertically transmitted to progeny through the egg, or from eliminating broodstock carriers and providing an overall reduction of pathogen presence at facilities. This approach has worked well for bacterial kidney disease (BKD). In addition to benefits associated with developing improved disease management strategies, identifying antigens that may be targeted for vaccine development will be important. If effective vaccine targets are identified then the long-term goal of developing a commercial CWD vaccine would provide a tool to prevent CWD at aquaculture facilities. Currently, such preventative measures do not exist and control relies on antibiotic use. Anticipated benefits associated with this project will include the availability of additional diagnostic tools (monoclonal antibodies and pathogen detection assays) for broodstock and/or egg culling to minimize CWD outbreaks, identification of potential vaccine candidates, and subsequent reduction of mortalities due to CWD. PROGRESS AND PRINCIPAL ACCOMPLISHMENTS: Funding for this project became available in February 2008, and a PhD student (Amy Long) was recruited in May 2008, a Postdoctoral Fellow (Rajesh Kumar) worked on this project from July 2008 to July 2009, and a PhD student (Karol Gliniewicz) has recently joined Dr. Call’s lab (2009). A program review of the project was conducted by Dr. Jerri Bartholomew in May 2010 and progress was reported to the WRAC board. The primary workgroup members were involved in this review and it offered an opportunity to confirm upcoming plans and discus results to date. A more formal workgroup meeting will be scheduled in November following the next round of sample collections at Troutlodge (currently scheduled for mid-October).


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    Objective 1: Identify potential vaccine candidates using comparative proteomic analysis of an attenuated strain of Flavobacterium psychrophilum and determine if crude cell lysate can be used as a subunit vaccine delivery vehicle in the absence of adjuvant. The original project proposal included efforts to develop and screen an IVIAT library to detect in vivo expressed antigens. As reported last year, while we were able to detect in vivo expressed proteins using large culture volumes, we were unable to obtain sufficient analytic sensitivity to differentiate in vivo expressed proteins from background when using a high-throughput format. Lacking this higher throughput, there was no practical means to screen the library. We received approval to change our focus for this component of the project to (1a) employ comparative proteomics to identify other candidate proteins based on differentially expressed proteins from a virulent strain (CSF259.93) and an attenuated strain (CSF259.93.B17), and (1b) to determine if subunit vaccine candidates can be screened using crude lysate from our Vibrio parahaemolyticus expression host. The intent of this second aim was to improve the efficiency of subunit testing and reduce overall cost of vaccine administration. Finally, time and resource permitting, we proposed to initiate work to determine why B17 is attenuated with the expectation that this would lead to a more rapid means to develop repeatable and stable attenuation in F. psychrophilum and other bacterial pathogens. Table 1. Differentially expressed proteins isolated from strains CSF259.93 and CSF259.93.B17 and identified by mass spectrophotometry. Mass (kDa) Reactive?a Putative function Differentially expressed in CSF259.93 FP1493 22.7 Yes Hypothetical protein; possible heme-binding lipoprotein; antigenic target of FL43 monoclonal antibody; appears to form multimers; currently undergoing vaccine trial YueD 27.8 TBD Benzil reductase FusA 79.3 Probable Elongation factor G Fpp2 100 Probable metaloprotease precursor DapB 26.3 TBD dihydropicolineate reductase FP1618 24 TBD hypothetical protein Differentially expressed in CSF259.93.B17 OmpA P60 49.4 Yes Outer membrane protein OmpA family P60 antigen Hsp70/DnaK 67.3 Yes Heat shock protein 70; chaperone RspA 66.4 TBD 30S ribosomal protein S1 FspA 21.3 Probable Flavo-specific protein antigen precursor FP0620 42.8 TBD hypothetical protein FP0192 128 TBD transcription-repair coupling factor a Yes = a protein that is recognized by antisera from convalescent rainbow trout as determined by our work or the published literature; Probable = protein isolated from region of gel that is reactive to antisera with minimal opportunity for contamination from other proteins; TBD = protein isolated from region of gel that has several adjacent immunoreactive proteins in the immediate vicinity. Probable and TBD proteins will be confirmed for immunoreactivity by western blot once recombinant proteins are produced (in progress). 1a. We have already presented data supporting the observation that strain CSF259.93.B17 (“B17”) is attenuated relative to the wild-type strain, CSF259.93, and that inoculation with strain B17 elicits a protective immune response (LaFrentz et al. 2008). Strain B17 was produced by


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    serial passage on agar plates containing increasing concentrations of the antibiotic rifampicin and during the past year we verified that an expected single-nucleotide mutation is present in the beta subunit of the RNA polymerase (RNAP). This point mutation is considered sufficient to convey resistance to rifampicin although we do not know why this change results in a fairly drastic alteration of expression profiles. A PhD dissertation proposal has been developed that will examine this latter question in more detail (see work planned for the final fiscal cycle). Two-dimensional gel electrophoresis experiments demonstrated that there are a number of differentially expressed proteins relative when B17 and CSF259.93 are compared (shown previously). Using mass-spectrophotometry we have now identified 12 proteins each that are differentially expressed by one strain or the other (Table 1) and we are in the process of expressing recombinant proteins and verifying the antigenicity of several of these proteins. Protein FP1493 has been identified in several published studies and it is the antigenic target of our monoclonal antibody (FL43) that we are using in diagnostic assays described in the aims below. Unlike many F. psychrophilum proteins, FP1493 is also relatively easy to express and purify. We are in the process of determining if immunization with FP1493 conveys protection against experimental challenge with F. psychrophilum. 1b. Based on earlier work, we rely heavily on a strain of Vibrio parahaemolyticus (strain NY-4) as our host bacterium for expression of recombinant F. psychrophilum proteins. V. parahaemolyticus usually works better than E. coli and we hypothesize that this is due to a closer match between codon usage for F. psychrophilum and V. parahaemolyticus compared with E. coli. Listonella angullarum, which is closely related to Vibrios, has been used successfully as a bacterin without addition Figure 1. Average percent mortality (±SEM) for purified proteins (first four of adjuvant. Consequently, bars), crude lysate from Vibrio parahaemolyticus strain NY-4, crude lysate from we hypothesized that we NY-4 with expressed proteins (mixed crude), and PBS + Freud's Complete could used crude lysate Adjuvant (PBS+FCA) control. There was no statistical difference in mortality from V. parahaemolyticus between groups (P= 0.07). A partial analysis of antibody titre showed very limited response to the the antigens tested (there was insufficient antisera as the vehicle for available for a complete analysis). The primary conclusion from this work is that delivering recombinant V. parahaemolyticus lysate does not stimulate a sufficient immunological protein in lieu of relatively response to provide protection from challenge with F. psychrophilum. At this expensive purification time we still require addition of an adjuvant, such as FCA, to successfully procedures. Our first two immunize trout for subunit vaccine evaluation. trial experiments indicated some support for this hypothesis, but our most recent trial indicates that delivery of recombinant protein in Vibrio lysate by itself is insufficient to provide protection following pathogen challenge (Fig. 1). Not only did we find no protective response, but a partial analysis of antisera


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    from convalescent fish indicated that lysate inoculated fish produced a minimal antibody titre. Consequently, we have rejected our hypothesis and will continue screening subunit vaccine candidates in conjunction with FCA. Our efforts for Objective 1 will henceforth focus on identification and testing of immunologically reactive antigens for vaccine development and proceed with experiments to determine the mechanism by which B17 is attenuated. Objective 2: Validate quantitative diagnostic assays (ELISA and ovarian fluid (membrane) filtration FAT). Optimization of the capture ELISA was completed during the past year. ImmunoPrecise Antibodies, Ltd. began producing horseradish peroxidase (HRP) conjugated MAb FL43 at high concentrations with a strong conjugation efficiency that facilitated switching back to an HRP based assay. Modification of wash steps, blocking steps, and sample preparation protocols allowed us to eliminate background and improve the detection limit. The detection limit of the capture ELISA is approximately 104 CFU ml-1 in both spiked kidney tissue and kidney samples from broodstock. We were able to successfully modify the capture ELISA to also allow testing of spleen samples. The detection limit for spiked spleen tissue is approximately 105 CFU ml-1. The MF-FAT does not allow effective quantification of bacterial load in the ovarian fluid; however, we can determine if fish are positive or negative by examining up to 150 fields per slide and marking samples as positive if at least one F. psychrophilum cell is observed. The positive-negative cutoff for the capture ELISA has been set as the average optical density (O.D.) of the negative control (unspiked tissue) plus two standard deviations. The specificity of the assay is 97.5% when this threshold is used (Barajas-Rojas et al. 1993). To estimate the number of CFU ml-1 in positive samples, a four parameter logistic equation was derived in GraphPad Prism for Windows v. 5.02 (Graphpad Software, San Diego, California) using the O.D. values from tissues spiked with a known concentration of F. psychrophilum. The best-fit curve of the known values was then used to interpolate CFU ml-1 for positive samples. Challenge Experiment A challenge experiment was initiate to relate infection levels and disease in fish injected with F. psychrophilum. Rainbow trout (average weight 39 g) reared in pathogen-free municipal water were split into two treatments: mock-infected and infected. Each treatment was done in triplicate with 5 fish per tank for a total of 15 fish per treatment. Fish were injected with 100 µl of either F. psychrophilum strain CSF 259-93 at a concentration of 2.7 x 108 CFU ml-1 or phosphate buffered saline. Three fish from each treatment were sampled prior to injection, and again at 7, 10, 14, and 21 days post-injection. Samples from kidney, liver, and spleen tissues were streaked on TYES plates, and plates monitored for bacterial growth. Kidney and spleen tissues were stored at -80°C until used in the capture ELISA. There were no mortalities during the challenge. The average O.D. for the samples is shown in Figure 2. Prior to injection, neither kidney nor spleen samples were positive by capture ELISA or by culture. At 7 days post-injection, the average kidney O.D. value was greater than the cutoff (0.094) while the average spleen O.D. was less than the cutoff (0.091) even though one of the


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    spleen samples was over the positive-negative threshold. At Day 10, one kidney sample from the treated group was greater than the cutoff but the average O.D. value for kidney was less than the cutoff. The O.D. value of this positive sample was 0.102 corresponding to 7.86 x 104 CFU ml-1. The average O.D. value for the spleen was also less than the cutoff on Day 10, and none of the individual samples were positive. At 14 and 21 days post-injection, none of the kidney or spleen samples were greater than the cutoff value indicating that fish had likely cleared the infection. F. psychrophilum was re-isolated from treatment fish on Day 7 but was not re-isolated throughout the remainder of the challenge. This includes the kidney sample from Day 10 that, according to the capture ELISA, had a concentration of 7.86 x 104 CFU ml-1. Based on these results, we can conclude that the capture ELISA is able to detect infections in fish exhibiting clinical symptoms of BCWD as well as in fish not exhibiting symptoms. Figure 2. Average O.D. values for (A) kidney and (B) spleen samples from fish injected with either F. psychrophilum or PBS. The range of the individual O.D. values is indicated by the vertical bars. Troutlodge Experiments In February 2010, 60 female broodstock were sampled at Troutlodge to evaluate broodstock infection levels. Eggs from each broodstock were kept in individual incubators while testing was underway. Ovarian fluid was assayed tested by nPCR, culture, and MF-FAT. In addition to


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    streaking on TYES plates, kidney and spleen samples were used in the capture ELISA. For MF- FAT samples, 150 fields were examined per slide and samples marked as positive if at least one F. psychrophilum cell was observed. In all, 100% of ovarian fluid samples were positive by the MF-FAT while only 6.67% were positive by nPCR. YPB isolates were confirmed as F. psychrophilum by nPCR. F. psychrophilum was re-isolated from 7 samples, 11.7%. Finally, three spleen samples were positive by ELISA while none of the kidney samples were positive (Fig. 3). Figure 3. Percentage of positive samples from Troutlodge in February 2010. Based on the results of these assays, five families were selected for the first round of experiments. The selected families were F54, F61, F70, F74, and F87 (Table 2). F54 was designated as the “low” infection family as it was negative by all assays except for the MF-FAT. The other four families were chosen because, in addition to positive MF-FAT, they either had positive ELISA results or F. psychrophilum was isolated from at least one sample. There were no samples that could have been classified as having a “high” level of infection. Eyed eggs were received from Troutlodge in mid-March. Upon arrival, eggs were surface disinfected using Ovadine® (PVP iodine) at a concentration of 100 ppm for ten minutes. Five eggs from each family were then sampled to determine if F. psychrophilum was present following disinfection. Fluid was withdrawn from each egg with a needle and syringe using aseptic techniques (Taylor 2004) and samples were pooled for each family. DNA was extracted from the fluid using a DNeasy Blood & Tissue DNA extraction kit (Qiagen, Carlsbad, CA) prior to nPCR testing.


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    Table 2. Summary of assay results for the five selected families. ELISA Ovarian Fluida Culture MF- Confirmed by Family Kid. Spleen CFU ml-1 nPCR FAT nPCR F54 - - . - + . F61 - + 1.21E+05 - + Kidney F70 - + 1.79E+06 - + - F74 - + 1.41E+06 - + . F87 - - . - + Spleen, OF a nPCR = nested PCR; MF-FAT = membrane filtration FAT Progeny were reared in separate flow through tanks at the University of Idaho wetlab facility until they reached an average weight of 0.5 g. During that time, families were sampled every week to check for F. psychrophilum. For the first seven weeks, fry were too small for necropsy; therefore, five fry from each family were pooled and disinfected in 400 ppm Ovadine® for 15 minutes (Brown et al. 1997). They were then rinsed five times in sterile water and then homogenized in an equal volume of sterile PBS. A sub-sample from the homogenate was plated on TYES media supplemented with 5 µg ml-1 of tobramycin (TYES-TB). At this concentration, F. psychrophilum can grow while tobramycin inhibits faster growing bacteria (Kumagai et al. 2004). DNA was extracted from the remaining homogenate with the DNeasy Blood & Tissue DNA extraction kit and used in nPCR. By Day 57, fry were large enough that kidney and spleen could be plated on TYES-TB and monitored for YPB growth. Table 3 lists both nPCR and culture results for this time period. On day 81, progeny were moved to the Aquaculture Research Institute’s (ARI) Coldwater Research Laboratory to begin stress experiments. In addition to the stress of transport, two stressors were used in this round of experiments; 1) gas supersaturation (chronic stress) and 2) gas supersaturation plus handling stress (acute stress). These particular stressors were chosen as previous studies have linked both to health problems in hatcheries and increased susceptibility to pathogens (Mesa et al. 2000, Dror et al. 2006). Chronic and acute treatments were done in triplicate for each family for a total of six tanks per family. Each tank had a volume of approximately 60 l and was stocked with 50 fry. All tanks were exposed to supersaturated water and acute treatment tanks were subjected to handling stress. Handling stress was done by collecting fish in a net and holding the net in the air for 20 seconds. This was done twice a day for 56 days. F. psychrophilum can be present without any clinical signs of disease so one fish was randomly sampled from each tank once a week for the duration of the experiment, 6 fish per family. Mortalities were examined to determine cause of death. For both random samples and mortalities, kidney, liver, and spleen were streaked on TYES-TB plates and sub-samples taken for nPCR.


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    Table 3. Summary of results from weekly monitoring of progeny. (-) = negative; (+) = positive; NS= no sample. Sampling Days Family Assaya 0b 9 16 23 29 36 44 51 57 64 72 nPCR + - + + + - - + NS NS NS F54 YPB - - - - - - + + - - - nPCR + + + + - - - - NS NS NS F61 YPB - - - - - - - - - - - nPCR + - + + + - - - NS NS NS F70 YPB - - - - - + + - - - - nPCR + + - + + - - - NS NS NS F74 YPB - - - - - + + + - - - nPCR + + - + - - - - NS NS NS F87 YPB - - - - - + - - - - - a nPCR = nested PCR; YPB = yellow pigmented bacteria b This time point represents samples taken from eyed eggs following disinfection. Nitrogen gas levels in the tanks ranged from 101-111% throughout the experiment. Fish exhibited signs of chronic stress including frayed fins, petechial hemorrhaging, scale loss, and overinflated swim bladders. In all, there were 27 mortalities during the challenge. The greatest number of mortalities occurred in F87. There were 10 mortalities in F87 compared to four mortalities each in F54, F70, and F74 and five mortalities in F61. The difference in mortalities was not significant when compared by a 1-way ANOVA. Results from the nPCR showed that F. psychrophilum was present in 29.6% of mortalities. Twenty-six percent of mortalities had YPB growth on TYES-TB plates but none of the re-isolated YPB were positive by nPCR. There was no bacterial growth on TYES-TB plates from the random samples. Nested PCR detected F. psychrophilum in all families throughout the experiment but there was not a significant difference in the number of times it was detected in each family. However, there was an increase in the proportion of positive fish (number of positive fish/total number of fish sampled per family) from each family throughout the experiment. At the beginning of the challenge, Day 82, the proportion of positive samples ranged from 0.17 to 1. By Day 138, the proportion of positive samples for all families was either 0.83 or 1 (Fig. 4). While we did not have a full-scale outbreak of BCWD, we did show an increase in F. psychrophilum in the population being studied. Determining the proportion of positive fish from the progeny monitoring portion is impossible as five fish from each family were pooled for one sample. We can, however, assume that if nPCR


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    was positive, then at least one of the five fish sampled was positive. The proportion of positives is then set as a minimum of 0.2 for all samples that were positive during this time period. F. psychrophilum was present in the eyed eggs upon arrival at UI indicating that the bacterium was passed from broodstock to progeny. Samples continued to test positive through Day 29. Between Day 36 and 51, there was only one positive sample, F54 on Day 51. It seems likely that as the progeny and the innate immune system developed, they were able to clear the infection to below detectable levels. Once the stress experiment was initiated, F. psychrophilum was detected in increasing proportion as time progressed. Figure 4. Proportion of samples positive for F. psychrophilum throughout entire trial. Dashed line represents the move from UI wetlab to the Coldwater Research Institute on Day 81. Asterisks denote sampling dates where only culture was done. Hatchery Studies As part of our ongoing efforts to validate the diagnostic assays, we once again solicited samples from regional hatcheries this year. Samples were collected from steelhead spawned at Wallowa Hatchery (WH) (Enterprise, OR) over the course of six weeks from March 2010 to May 2010. In addition, we were able to obtain samples collected in 2003 from Lower Elwha and Skookum Creek hatcheries. We chose these samples as they were collected the same year and from the same facilities as the samples used when developing the original capture ELISA (Lindstrom et al. 2009). Sixty kidney and 59 spleen samples from WH were tested by the capture ELISA. Approximately 23% of kidney samples and 8% of spleen samples were positive. The estimated CFU ml-1 for kidney samples ranged from 4.92 x 104 to 2.34 x 106. All positive spleen samples had low O.D. values that were below the best-fit line for the standard curve and as such, CFU ml-1 could not be estimated effectively. F. psychrophilum was isolated from kidney, spleen, and ovarian fluid


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    samples as well. Outbreaks of F. psychrophilum have been noted in progeny from these broodstock. The coho salmon samples collected in 2003 had a range of 3.85 x 104 to 9.09 x 105 CFU ml-1. Seven of the 20 samples collected at the Lower Elwha hatchery were positive while all of the samples from Skookum Creek were positive. Our results agree with those published by Lindstrom et al. (2009) in that 100% of those samples were positive for F. psychrophilum. Objective 3: Based on results from objective 2: Develop other assays (e.g. real-time quantitative PCR) for quantification of infection in ovarian fluid. The amino acid sequence for the antigen recognized by MAb FL43 was sequenced this year by the Call Lab. Using the amino acid sequence, we were able to search the F. psychrophilum genome for the corresponding gene. The gene encoding FP1493 is approximately 645 nucleotides long, and is present in only one copy in the F. psychrophilum genome. A BLAST search of the sequence shows that the only the first ~200 nt are unique to F. psychrophilum and the remainder of the gene is similar to Flavobacterium johnsoniae. While F. johnsoniae is not a fish pathogen, it is ubiquitous in soil and freshwater. To avoid the possibility of false positives, probe and primer development for the qPCR assay were limited to the first 200 nt of FP1493. As the reagents for the Sybr Green qPCR assay are generally less expensive than those used in a TaqMan assay, we focused first on developing a Sybr Green qPCR protocol with the assumption that we could develop a TaqMan assay if the Sybr Green was unsuccessful. A section of FP1493 containing the sequence of interest was cloned into a plasmid using the TOPO® Cloning Kit (Invitrogen, Carlsbad, CA). The plasmid was then sequenced to ensure cloning was successful. Once confirmed, the plasmid DNA was used as template in the Sybr Green qPCR. We were able to successfully optimize the Sybr Green assay. Primer efficiency was 106% with an R2 value of 0.998. DNA extracted from Aeromonas salmonicida was run in the same assay and there was no amplification. Objective 4: Develop an integrated outreach program to meet stakeholder needs. Outreach activities have resulted in two articles in Waterlines describing this WRAC project. Project deliverables resulting from this work so far include the commercialization of monoclonal antibody FL43 through Immunoprecise Antibodies, Inc. Labeled antibodies are now being sold to researchers and aquaculture facilities interested in improved diagnostics for BCWD. Protocols for ELISA and FAT assays developed in our lab were provided to Immunoprecise and are included when antibody is sold.


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    USEFULNESS OF FINDINGS: Progress in diagnostic improvements is moving forward very well and should result in correlation of assays to disease risk in progeny from infected broodstock. Such finding would increase the need for labs to purchase the commercialized antibody and utilize assay protocols. Vaccine work has shifted from original objectives, but as we begin to define attenuation mechanisms this could lead to more efficient ways to create vaccines strains for F. psychrophilum and possibly other fish bacterial pathogens. WORK PLANNED FOR NEXT YEAR: Objective 1: Identify and test potential vaccine candidates based on comparative proteomic analysis of CSF259.93 and CSF259.93.B17 and determine the mechanism of B17 attenuation. We have completed the proteomic analysis and with the exception of some material currently in process, we do not plan to identify any additional differentially expressed proteins. At this time we will proceed with expression of recombinant proteins and verify antigenicity by western blot using antisera from convalescent rainbow trout. Antigenic proteins will enter our testing “pipeline” that involves immunizing trout followed by challenge with strain CSF259.93 (e.g., Sudheesh et al. 2007). Importantly, each immunization trial requires approximately three months to execute and there are usually only sufficient fish and tanks available to complete one immunization trial at a time. Thus, in the time remaining in this project we will not be able to assess every protein identified in this study. Instead, we will focus on proteins that are immunologically reactive and that are probable virulence factors such as the Fpp2 (Table 1) although this will depend in part on our ability to produce a sufficient mass of the protein of interest. The expected deliverable for this segment of our project is a manuscript describing the differentially expressed proteins and, if successful, a manuscript describing identification of a vaccine candidate that induces significant protective immunity. Our working hypothesis is that attenuation of B17 results from altered global transcriptional regulation as evidenced by a significant change in protein expression pattern relative to the pathogenic parental strain CSF259-93. Altered expression is probably a result of a point mutation in the beta subunit (rpoB) of the RNA polymerase and this year we have confirmed that one of the expected mutations is present in B17. We predict that the pattern of altered protein expression is repeatable, predictive of attenuation, and can be engineered independently from rifampicin passage. The mechanism underlying altered protein expression most likely involves a change in how the RNA polymerase binds promoter sequences or a change in how the RNA polymerase interacts with key transcriptional factors (sigma factors). There is insufficient time (1 year) and resources remaining for this project to fully test both mechanisms. Nevertheless, we are well positioned to determine if promoter sequence binding plays a role in this process. That is, we will determine if mutations in the rpoB affect the efficiency of promoter binding using the differentially regulated genes as “bait” for this assay. This will be followed by introducing an identical mutation into a pathogenic strain of F. psychrophilum to recapitulate attenuation. The rationale for this series of experiments is that we will identify the specific mechanism involved in rifampicin attenuation (currently a knowledge gap), which will provide the opportunity to generate a genetically engineered and potent live-attenuated strain that will also be unlikely to


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    revert to virulence. Knowledge of the precise mechanism of rifampicin attenuation may also offer a possibility of creating of attenuated strains of other bacterial pathogens Determine if mutations in rpoB affect efficiency of promoter binding. The first part of this work involves a detailed analysis of differentially expressed proteins (Table 1) to identify their promoters and transcriptional binding sites. We will then use the promoter sequences to construct oligonucleotides that will be employed in electrophoretic mobility shift and in vitro transcription experiments with purified native and rifampicin-resistant RNAPs to determine if binding affinity varies. To obtain pure RNA polymerases (“holoenzymes”), whole-cell lysates of CSF259.93 and B17 strains will be used for immunoaffinity purification (Thompson et al., 1992) of the native and mutant enzymes, respectively. Lysates will be processed through columns loaded with 8RB13 (anti-RpoB) and 8RC8 (anti-RpoC) “polyol-responsive” cross-reactive monoclonal antibodies against RNAP and the enzyme will be eluted under very gentle conditions. These antibodies have been effective for purification of RNAPs from various bacterial species (Bergendahl et al., 2003) and we have already acquired the necessary antibodies and have demonstrated that they recognize the RNAP from F. psychrophilum (western blot data not shown). A non-specific in vitro transcription assay will be used to confirm that the purified holoenzyme is functional. This assay involves assembling a transcription buffer that includes the necessary components for transcription including the RNAP of interest, a biotinylated–UTP, and genomic DNA template from F. psychrophilum. After a simple 30-min incubation period, we will size separate any RNA transcripts using gel electrophoresis and detect the labeled products using a conventional colorimetric assay. An electrophoretic mobility shift assay (EMSA) will be used to assay changes in the ability of the wild-type and mutant RNAPs to bind the promoter regions of the differentially expressed proteins from Table 1. This involves mixing the purified RNAPs with double-stranded synthetic oligonucleotides (75 nt-long) identical to the promoter sequences from Table 1 proteins. A 75 nt- long promoterless oligonucleotide will be used as a negative control. After the protein and DNA are allowed to bind, this complex is examined by electrophoresis. If binding occurs, migration through the gel will be retarded relative to the oligonucleotide–only control. We expect to see differences in absolute binding (on-off) or in binding affinity as assessed by differences in the concentration of RNAP required to detect retarded migration in the EMSA. We will also assess affinity using promoter-specific in vitro transcription. This is similar to the non-specific transcriptional assay described above, except that specific promoters for proteins in Table 1 will be used. Densiometry will be used to quantify differences in transcription products, but we can also use quantitative PCR (either Sybr Green or Taqman based) to measure the number of transcripts produced form this experiment. Introduce mutated rpoB into F. psychrophilum THC02/90 strain and determine if this replicates changes in transcription profiles and attenuation. F. psychrophilum is difficult to genetically manipulate by homologous recombination, although molecular methods were recently established (McBride and Baker, 1996; Alvarez et al., 2003) and we have acquired the necessary reagents from Dr. Mark McBride. A second complicating factor is that our pathogenic strain,


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    CSF259.93, belongs to a genetic lineage of F. psychrophilum that cannot be genetically manipulated using conventional tools; presumably due to restriction enzyme incompatibilities (Alvarez et al., 2003; Soule et al., 2005; Chen et al., 2008). Therefore, we will instead use the THC02/90 strain of F. psychrophilum that has been successfully employed for genetic manipulations (Alvarez et al., 2006) and we have shown that this strain is virulent in our fish challenge model (Call et al., unpub. data). If our hypothesis that attenuation results from mutations in RpoB of RNA polymerase is correct, then we expect that introduction of the mutant version of rpoB into F. psychrophilum THC02/90 strain will reproduce the abnormal gene expression resulting in full attenuation. Full allelic exchange has not been successful in F. psychrophilum, but we can create gene knockouts and use a complementation vector to restore function with a mutated sequence that has a native promoter (Rhodes et al., 2010). We will introduce the mutant rpoB by homologous recombination through application of a bacteroidete suicide vector pLYL03 (Rhodes et al., 2010). This involves first transforming THC02/90 with a Bacteroides-Flavobacterium suicide vector-pLYL03 (AmpR/ErmR) that harbors a kanR gene (kanamycin resistance) that is flanked by sequences homologous to regions of the native rpoB gene. Introduction of this vector should lead to disruption of rpoB with the antibiotic cassette by a single recombination event. The second plasmid will be a low copy Flavobacterium shuttle plasmid pCP29 (AmpR/CfxR/ErmR) carrying a mutant version of rpoB. Because only one copy of rpoB is present on the bacterial chromosome, incorporation of kanR should disrupt the native rpoB gene leaving a functional copy of mutated rpoB gene on pCP29. Double transformants will be selected on TYES agar containing kanamycin and cefoxitin. Presence of the pCP29 will be confirmed by plasmid miniprep and kanR integration by PCR with specific primers for junction regions. Assuming that the engineered strain is not significantly harmed (slow growing), it will be a stable mutation because loss of the expression vector will lead to non-viable cells. Given a stable mutant strain, we can proceed to test both alterations in protein expression and test for attenuation using our fish challenge model. Importantly, as a backup procedure we have already begun passaging THC02/90 on rifampicin plates as an alternative means to generate the rpoB mutation. With this conventional selection technique we can still determine if altered protein expression and attenuation is a repeatable outcome although we will have less control over other spontaneous changes that occur during the selection process (this can take weeks to achieve resistant strains). If successful, we anticipate three manuscripts from objective 1. One paper will describe the altered transcriptional profile of B17 relative to the virulent wild-type strain. A second paper will describe results from the immunization trials, and hopefully will describe a protein that conveys significant protective immunity. A third paper will describe the mechanism by which rifampicin selection producing attenuation. Objective 2: Validate quantitative diagnostic assays (ELISA and ovarian fluid (membrane) filtration FAT). Sampling for the second round of experiments will be done at Troutlodge in late Fall 2010. As before, kidney, spleen, and ovarian fluid samples from 60 fish will be collected and tested using the capture ELISA, MF-FAT, nPCR, culture, and qPCR. Five families with a range of infection levels will be selected and eyed eggs sent to UI. Stressors for this round of experiments have yet to be decided upon; however, we will most likely continue to use a chronic stressor in addition to


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    an acute stress such as a one-time low dissolved oxygen level stress. Depending on the size of progeny fish, we may collect anterior kidney samples and attempt to quantify the levels of F. psychrophilum present. Susceptibility of the progeny when directly challenged with F. psychrophilum will also be analyzed if we are unable to induce a disease outbreak in the stress experiments. Hatchery Studies Collection of broodstock samples from hatcheries in the region will also continue in the upcoming year. Beginning in November 2010, we will receive coho salmon broodstock samples from Skookum Creek Fish Hatchery once a week for six weeks. We chose this hatchery as 100% of the samples from 2003 were positive and severe outbreaks of BCWD are common at this facility. As before, eggs will be monitored by hatchery personnel and any BCWD outbreaks reported. In addition, Oregon Department of Fish and Wildlife has agreed to continue to provide steelhead samples from WH and track outbreaks in progeny. Multi-year collection from one hatchery with a history of BCWD will help as we evaluate the relationship of infection levels in broodstock to outbreaks in progeny and validity of our assays. Objective 3: Based on results from objective 2: Develop other assays (e.g. real-time quantitative PCR) for quantification of infection in ovarian fluid. Work on the qPCR will continue in the upcoming year. Our first task will be confirming that the primers are specific to F. psychrophilum and do not react to any other Flavobacterium species. Evaluation of the sensitivity of the qPCR will also need to be done. Once that is done, we will begin analyzing DNA from ovarian fluid samples collected during the past two years. These samples have been stored according to manufacturer's directions and should still be viable. Results from the qPCR will be compared to the nPCR, culture, and MF-FAT results. We have also begun working with Infoscitex, Inc. on developing new diagnostic assays. Infoscitex was recently awarded a SBIR grant from USDA to develop an aptamer assay for F. psychrophilum. Aptamers are single-stranded DNA or RNA oligonucleotides that are only tens of nucleotides long. There are several benefits to using aptamers in place of antibodies. The first is that aptamers are able to bind viruses, proteins, and small molecules. The second benefit of aptamers is that they are synthetically produced so a large batch can be made in a matter of days and have a long shelf life. Phase I of the grant has just gotten underway. The objective for this phase is the production of an aptamer for F. psychrophilum. Infoscitex is currently working on this using purified FP1493 protein. Once an aptamer has been produced that has demonstrated high sensitivity and selectivity to F. psychrophilum, ELISA and qPCR assays will be developed. Aptamers produced by the technology that Infoscitex will use have detected as low as 10-16 g of target material. If a successful aptamer assay is developed, we may be able to lower our detection limit of F. psychrophilum from 104 CFU ml-1 by capture ELISA to a single CFU per sample. Objective 4: Develop an integrated outreach program to meet stakeholder needs. We will continue to increase awareness of project and research progress through popular press articles in aquaculture newsletters and presentations at aquaculture and fish health meetings. The


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    integrated approach will follow established guidelines in relation to each objective (see original proposal). Based on project deliverables, we will evaluate the need for workshops this coming year and begin development of a draft WRAC Extension publication. Based on project deliverables, evaluate need for workshops and develop a draft WRAC Extension publication. We will also continue to work with Immunoprecise to inform stakeholders of the availability of improved diagnostic tools. References: Alvarez J, Menendez A, Guijarro JA. A mutant in one of two exbD loci of a TonB system in Flavobacterium psychrophilum shows attenuated virulence and confers protection against cold water disease. Microbiology. 2008 154:1144-51. Alvarez B, Secades P, Prieto M, Guijarro JA. (2006) A mutation in Flavobacterium psychrophilum tlpB inhibits gliding motility and induces biofilm formation. Appl. Environ. Microbiol. 72, (6): 4044-4053 Alvarez B, Secades P, McBride MJ, Guijarro JA. Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum. Applied Environmental Microbiology. 2004 70:581-7 Barajas-Rojas JA, Riemann HP, Franti CE (1993) Notes about determining the cut-off value in enzyme-linked immunosorbent assay (ELISA). Preventive Veterinary Medicine 15:231-233 Bergendahl V, Thompson NE, Foley KM, Olson BM and Burgess RR. (2003) A cross-reactive polyol-responsive monoclonal antibody useful for isolation of core RNA polymerase from many bacterial species. Protein Exp Purif. 31: 155–160 Brown LL, Cox W, Levinel RP (1997) Evidence that the causal agent of bacterial cold-water disease Flavobacterium psychrophilum is transmitted within salmonid eggs. Diseases of Aquatic Organisms 29:213-218 Chen, J, MA Davis, SE LaPatra, K Cain, K Snekvik, and DR Call. 2008. Genetic diversity of Flavobacterium psychrophilum recovered from commercially raised rainbow trout and spawning Coho salmon. Journal of Fish Diseases 31:765-773 Dror M, Sinyakov MS, Okun E, Dym M, Sredni B, Avtalion RR (2006) Experimental handling stress as infection-facilitating factor for the goldfish ulcerative disease. Veterinary Immunology and Immunopathology 109:279-287 Grainger DC, Busby SJ. Methods for studying global patterns of DNA binding by bacterial transcription factors and RNA polymerase. Biochem Soc Trans. 2008 36:754-7. Kumagai A, Nakayasu C, Oseko N (2004) Effect of tobramycin supplementation to medium on isolation of Flavobacterium psychrophilum from ayu Plecoglossus altivelis. Fish Pathology 39:75-78


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    LaFrentz, B.R., LaPatra, S.E., Call, D.R., Wiens, G.D. and Cain, K.D. Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media. Diseases of Aquatic Organisms 87: 171-182 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 (2008) 5582–5589 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 LaFrentz, B.R., LaPatra, S.E., Jones, G.R. and Cain, K.D. 2003. Passive immunization of rainbow trout (Oncorhynchus mykiss) to Flavobacterium psychrophilum, the causative agent of coldwater disease and rainbow trout fry syndrome. Journal of Fish Diseases 26, 377-384 LaFrentz, B.R., LaPatra, S.E., Jones, G.R., Congleton, J.L., Sun, B. and Cain, K.D. 2002. Characterization of serum and mucosal antibody responses and relative percent survival in rainbow trout (Oncorhynchus mykiss) following immunization and challenge with Flavobacterium psychrophilum. Journal of Fish Diseases. 25, 703-713. Lindstrom, N.M., Call, D.R., House, M.L., Moffitt, C.M., and Cain, K.D. 2009. A quantitative enzyme-linked immunosorbent assay (ELISA) and filtration-based fluorescent antibody test (FAT) as potential tools to screen broodstock for Flavobacterium psychrophilum infection. Journal of Aquatic Animal Health 21(1): p. 43-56 Liu Y, Wei L, Batzoglou S, Brutlag DL, Liu JS, Liu SS. A suite of web-based programs to search for transcriptional regulatory motifs. Nucleic Acids Res 2004 32:34-7 McBride MJ and Baker SA. (1996) Development of techniques to genetically manipulate members of the genera Cytophaga, Flavobacterium, Flexibacter and Sporocytophaga. Appl. Environ. Microbiol. 62: 3017-3022 Mesa MG, Maule AG, Schreck CB (2000) Interaction of infection with Renibacterium salmoninarum and physical stress in juvenile chinook salmon: Physiological responses, disease progression, and mortality. Transactions of the American Fisheries Society 129:158-173 Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001 29:e45 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): p. 521-34


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    Rhodes RG, Samarasam MN, Shrivastava A, van Baaren JM, Pochiraju S, Bollampalli S and McBride MJ. (2010) Flavobacterium johnsoniae gldN and gldO Are Partially Redundant Genes Required for Gliding Motility and Surface Localization of SprB. J. Bacteriol. 192 (5): 1201-1211 Soule M, LaFrentz S, Cain K, LaPatra S, Call DR. Polymorphisms in 16S rRNA genes of Flavobacterium psychrophilum correlate with elastin hydrolysis and tetracycline resistance. Dis Aquat Organ 2005 65:209-16 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 Taylor PW (2004) Detection of Flavobacterium psychrophilum in eggs and sexual fluids of Pacific salmonids by a polymerase chain reaction assay: Implications for vertical transmission of bacterial coldwater disease. Journal of Aquatic Animal Health 16:104-108 Thompson NE, Hager DA and Burgess RR, (1992) Isolation and characterization of a polyol- responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase, Biochemistry 31: 7003–7008 Zhou X, Shah DH, Konkel ME, Call DR. Type III secretion system 1 genes in Vibrio parahaemolyticus are positively regulated by ExsA and negatively regulated by ExsD. Mol Microbiol. 2008 69:747-64 IMPACTS: The primary impact is the commercialization of monoclonal antibody FL43 through Immunoprecise Antibodies, Inc. This is now being sold to research labs and/or aquaculture companies in the un-conjugated form or conjugated to FITC or HRP. Protocols for the capture ELISA and FAT have been distributed to fish health labs in the region. Furthermore, we have provided these protocols to ImmunoPrecise to be distributed to customers when they purchase FL43. SUPPORT: WRAC-USDA Other Total Year University Industry Other Total Funding Federal Support 2008 $81,555.00 $81,555.00 2009 $80,043.00 $80,043.00 2010 $81,637.00 $81,637.00 $81,639 $81,639 2011 (approved 10/09) Total $324,874.00


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    PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: Refereed publications: LaFrentz, BR, SE LaPatra, DR Call, GD Wiens, and KD Cain. 2009. Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media. Diseases of Aquatic Organisms 87:171-182. PMID: 20099411. 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 21:43-56. PMID: 19485125. 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): p. 521-34 Plant, KP, SE LaPatra, DR Call, and KD Cain. In review. Immunization of rainbow trout (Oncorhynchus mykiss) with Flavobacterium psychrophilum proteins elongation factor-Tu, SufB Fe-S assembly protein and ATP synthaseb. General articles: Cain, K.D. 2009. Strategies for Control and Prevention of Coldwater Disease. Waterlines newsletter 15 (1): p. 18-20 Cain, K.D. Call, D.R, and Snekvik, K.R. 2010. A tail of two diseases (Coldwater disease and Strawberry disease research) Waterlines newsletter 16 (1): p. 10-11 Presentations: Gliniewicz, Cain, Snekvik, and Call, “The role of rpoB in the attenuation of Flavobacterium psychrophilum after passage with rifampicin” Poster presented at the 10th Annual College of Veterinary Medicine Research Symposium, October 14, 2009. Long, A., Call, D.R., and Cain, K.D. 2009. Comparison of diagnostic techniques for detection of Flavobacterium psychrophilum in ovarian fluid. Talk presented at the 50th Western Fish Disease Workshop and AFS Fish Health Section Annual Meeting. Park City, Utah. June 7-10. Gliniewicz, K, K Snekvik, K Cain, S LaPatra, and D Call. Assessing the immune-protective potential of FP1493 against coldwater disease in rainbow trout. Poster presented at American Society for Microbiology general meeting, May 2010, San Diego, CA. Lanier, A, R Kumar, S LaPatra, K Gliniewicz, K Snekvik, K Cain, D Shah, and D Call. Production of recombinant in vivo induced proteins of Flavobacterium psychrophilum for


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    development of a cold water disease vaccine for rainbow trout. Poster presented at the WSU Showcase, March 2010, Pullman, WA. Long, A., Call, D.R., and Cain, K.D. 2010. Use of Diagnostic Assays to Screen Rainbow Trout (Oncorhynchus mykiss) Broodstock for Flavobacterium psychrophilum. Talk presented at the 6th International Symposium for Aquatic Animal Health and AFS Fish Health Section Annual Meeting. Tampa, Florida. September 5-9. SUBMITTED BY:______________________________________September 8, 2010____ Title: (Work Group Chair or PI) Date September 13, 2010 APPROVED: _____________________________________________________ Technical Advisor (if Chair’s report) Date


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