WRAC ANNUAL PROGRESS REPORT – Year 2

PART I: Summary PROJECT TITLE: Coldwater disease prevention and control through vaccine

development and diagnostic improvements

REPORT GIVEN IN YEAR: 2009 REPORTING PERIOD: September 1, 2008 – August 31, 2009 (2nd 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 (approved 10/08) Fourth Year Request: $81,639

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 Student: Amy Long University of Idaho 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 in vivo-induced antigen technology. • 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 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 and a Postdoctoral Fellow (Rajesh Kumar) worked on this project from July 2008 to July 2009 at the University of Idaho (Cain) and Washington State University (Call), respectively. A workgroup meeting (conference call) was held in July 08 and another will be held in late September to discuss results to-date and upcoming needs for next year. Objective 1: Identify potential vaccine candidates using in vivo-induced antigen technology. The IVIAT library is designed to detect in vivo expressed antigens and involves generating a genome-wide expression library that is screened with antibodies from convalescent rainbow trout

serum. The antibodies used in the screening process are absorbed against in vitro grown F. psychrophilum to isolate antibodies that are specific for in vivo expressed proteins. For this system to work we require (1) successful expression of proteins from F. psychrophilum, (2) suitable convalescent antisera, (3) a suitable positive control, and (4) sufficient analytic sensitivity, which is a function of total protein expressed and reactivity/affinity of the convalescent antisera. To date we have demonstrated that we can detect our positive control protein (a putative in vivo-expressed protein, ACP21) using an experimental format that is compatible with library screening. This was an important development, but subsequent efforts yielded mixed results with significant obstacles due to poor analytic sensitivity. We might be able to increase analytic sensitivity by moving the library from an E. coli expression host to a Vibrio parahaemolyticus expression host based on findings in our lab, but preliminary work indicates that we will continue to encounter high variance between screening experiments and unreliable detection of our positive control protein even with a V. parahaemolyticus expression host. We surmise that after absorption against in vitro grown F. psychrophilum, the remaining antibody titer is simply too low for reliable detection of expressed proteins in a 96-well format (this can be improved dramatically with larger volume culture, but this alternative is not practical for library screening). Based on our efforts to date, we propose an alteration in our experimental plan. We recently developed a strain of F. psychrophilum (strain 259-93B.17) that has been completely attenuated as demonstrated by injection challenge trials in rainbow trout. Furthermore, we have shown that immunization with strain B.17 is sufficient to confer significant protection against F. psychrophilum challenge. Preliminary characterization of B.17 relative to the parent strain CSF259-93 shows that there are a number of differentially expressed proteins that are of immediate interest with respect to development of vaccine targets. These results also suggest that attenuation is due to alteration of global transcriptional regulation and that further investigation into the mechanism of attenuation could yield valuable information about how to develop attenuated bacteria more efficiently and with a higher degree of stability. Finally, we have preliminary data showing that recombinant proteins expressed in V. parahaemolyticus can be delivered to fish as a crude lysate preparation and this strategy appears to induce a protective immune response. This latter finding supports our earlier expectations and adds further credibility to our original strategy of using Vibrio as the expression host. The ability to use crude lysates to test vaccine candidates represents a large cost savings and our data also suggest that Vibrio could be used as a low-cost, alternative adjuvant for testing vaccine candidates. Objective 2: Validate quantitative diagnostic assays (ELISA and ovarian fluid filtration FAT). We have continued to work on optimizing the ELISA and the membrane filtration FAT (MF-FAT) for detecting F. psychrophilum in kidney tissue and ovarian fluid. Optimization of the MF-FAT has been successful, and the detection limit of the assay has been calculated. By spiking sterile filtered ovarian fluid with known concentrations of F. psychrophilum CSF 259-93, we were able to set the detection limit at 8.8 x 103 CFU ml-1. Once preliminary optimization of the MF-FAT was completed, ovarian fluid samples from rainbow trout and steelhead broodstock reared at two different hatchery facilities were analyzed. Of the 118 samples collected, 38 were

determined positive for F. psychrophilum infection. We have also developed an indirect FAT (IFAT) using MAb FL43 conjugated to Alexa Fluor® 488 that can readily detect F. psychrophilum in tissue imprints as well as swabs from whole fish homogenates. Optimization of the sandwich ELISA for detection of F. psychrophilum in kidney tissue has been more complicated. The assay was redesigned and the biotin-streptavidin system incorporated in an attempt to increase sensitivity; however, sensitivity has not increased as desired. With biotin labeled monoclonal antibody FL43, the assay is able to detect 106 CFU ml-1 in kidney samples spiked with F. psychrophilum. Our original work (Lindstrom et al., 2009) reported what appeared to be greater sensitivity (~ 2 x 103 CFU ml-1). Upon further evaluation, it is appears that this reported difference may be due to methods used in bacterial enumeration rather than a true loss in sensitivity. We are confirming this, but we know that strains of F. psychrophilum have a tendency to auto-agglutinate and therefore original plate counts may have underestimated the number of starting bacterial. Experiments to confirm this and find the best method for enumerating bacterial populations are underway. To further evaluate assay sensitivity on infected fish, we have recently initiated a disease challenge experiment in which juvenile rainbow trout have been infected with two different doses of F. psychrophilum, high and low, and samples are being taken from the fish throughout the challenge to provide data on how ELISA measurements vary over the course of a disease challenge. Objective 3: Based on results from objective 2: Develop other assays (e.g. real-time quantitative PCR) for quantification of infection in ovarian fluid. No progress to report. Objective 4: Develop an integrated outreach program to meet stakeholder needs. Outreach activities have resulted in of an article describing this project to WRAC that was published in Waterlines [Vol 15(1)].

USEFULNESS OF FINDINGS: Findings thus far, along with previous development of the monoclonal antibody (FL43) has led to a license agreement between the University of Idaho and an antibody company (Immunoprecise) out of Vancouver, BC. This company has now produced FL43 and it is available commercially to diagnostic laboratories, research labs, and others wishing to test for CWD. Based on further results from this WRAC project, Immunoprecise will develop ELISA and FAT kits for the industry and research labs. WORK PLANNED FOR NEXT YEAR: Objective 1: Identify potential vaccine candidates using in vivo-induced antigen technology. Transitioning the IVIAT library to a Vibrio parahaemolyticus expression host library presents a potential solution to the problems of insufficient analytic sensitivity, but preliminary work on

this alternative indicates that we will still encounter a low signal to noise ratio because we will still need concentrated (1:50) antisera for antigen detection. We propose to redirect this portion of the project to instead focus on two aims: (1) Characterize the differentially expressed proteins between the virulent strain CSF259-93 and the attenuated daughter strain B.17. Proteins exclusive to strain CSF259-93 represent potential virulence factors so identification of these proteins will further our knowledge of F. psychrophilum pathogenicity while providing additional targets for subunit vaccine development. (2) Determine the mechanism responsible for attenuation of the B.17 strain. Our working hypothesis is that attenuation results from altered global transcriptional regulation and that this pattern of change is repeatable, predictive of attenuation, and can be engineered independently from rifampicin passage. To test this hypothesis and the alternative mechanisms (expression differences due solely to changes in RNA polymerase activity or due to changes in environmental sensing pathways) we will complete our proteomic and sequence analysis to identify transcriptional binding sites that we will then use as “bait” to capture and identify transcriptional regulators involved in this process. We will use parallel rifampicin passage experiments to determine if the same pattern of transcriptional alterations is a repeatable phenomenon. Proteomic and sequence analysis will be used to confirm changes in the passaged strains and we will determine if strains with similar characteristics to 259-93B.17 are attenuated. Finally, if resources permit we will generate deletion mutants to confirm their role in altered transcriptional regulation and attenuation. The rationale for this work is that we will know the precise mechanism involved in attenuation, which means that we will have the opportunity to design a more robust attenuated strain (less likely to revert to virulence), but more importantly, we will identify a predictable means for engineering attenuated strains with the clear implications that this procedure can be used to create attenuated strains of other important bacterial pathogens. Objective 2: Validate quantitative diagnostic assays (ELISA and ovarian fluid filtration FAT) In the upcoming year, we will use the ELISA and MF-FAT to screen broodstock reared at Troutlodge, Inc. for F. psychrophilum. Beginning this fall and continuing throughout the year, kidney tissue and ovarian fluid will be collected from up to 60 3-year-old broodfish. Samples will be transported to UI, analyzed using culture techniques and either ELISA (kidney tissue) or MF-FAT (ovarian fluid). While the samples are being tested at UI, fertilized eggs from the sampled broodstock will be kept in isolated incubators at Troutlodge. ELISA, MF-FAT, and culture results will be used to select progeny from up to 10 broodstock showing either high or low levels of F. psychrophilum infection. When fish reach the eyed stage, they will be shipped to UI for further experiments and grouped according to broodstock infection level. Fish will be reared to approximately 0.5 g at which point they will be separated and stocked in triplicate into 20 l tanks. Fish will then be subjected to a controlled stressor for a minimum of 28 days in an attempt to induce a F. psychrophilum outbreak. Random sampling of the fish will be done throughout the experiment to determine if F. psychrophilum is present. All mortalities will be examined for clinical symptoms of coldwater disease and attempts will be made to isolate F. psychrophilum from the kidney, liver, and spleen. Additionally, kidney and spleen imprints will be collected and screened for the presence of the bacterium using IFAT. These experiments will allow us to correlate the level of infection in the parent at time of spawning to the risk of a

disease outbreak in the progeny. Fish will be examined for F. psychrophilum before the start of every experiment. Progeny from broodstock which are negative for F. psychrophilum will be included in all trials to serve as negative controls. Objective 3: Based on results from objective 2: Develop other assays (e.g. real-time quantitative PCR) for quantification of infection in ovarian fluid. Development of a real-time quantitative PCR (qPCR) that can determine F. psychrophilum concentrations in ovarian fluid is essential as it will allow for non-lethal sampling of fish. Additionally, real-time qPCR is a quantitative and precise method for determining concentration of bacterial cells in both tissue and culture. In the upcoming year, we will begin to develop a real-time qPCR assay that can detect F. psychrophilum in both ovarian fluid and kidney tissue. We intend to develop a probe that is specific to the gene that encodes the antigen that is recognized by monoclonal antibody FL43. The sequence of this gene is currently unknown; however, Dr. Call's lab is close to elucidating the sequence. Once that sequence is known and assuming that is it is specific to F. psychrophilum and does not share homology with any other genes, we will begin developing protocols for extracting bacterial DNA from samples and optimizing the assay. Ovarian fluid and kidney tissue sampled from broodstock at Troutlodge and used for Objective 2 will be preserved at -80°C for use in the real-time qPCR assay. Additionally, both ovarian fluid and kidney tissue samples have been collected from Lower Elwha hatchery and Dworshak National Fish Hatchery and can be tested by the real-time qPCR. These samples can be used to evaluate F. psychrophilum levels at different facilities. Objective 4: Develop an integrated outreach program to meet stakeholder needs. Increase awareness of project and research progress through popular press articles in aquaculture newsletters and presentations at aquaculture meetings. IMPACTS: Currently, the primary impact is the recent commercial availability of monoclonal antibody FL43. The agreement with Immunoprecise and their involvement will be pivotal in implementing findings from the diagnostic component of this research. SUPPORT:

Year WRAC-USDA Funding University Industry Other

Federal Other Total Total Support

2008 $81,555.00 $81,555.00 2009 $80,043.00 $80,043.00

2010 $81,637.00 (approved 10/08) $81,637.00

Total $81,555.00 $243,235.00

PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: Refereed publications: 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 (In Press) 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 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 General articles: Cain, K.D. 2009. Strategies for Control and Prevention of Coldwater Disease. Waterlines newsletter 15 (1): p. 18-20 Presentations: 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.

SUBMITTED BY: ____________________________________September 8, 2009____ Title: (Work Group Chair or PI) Date

APPROVED: _____ __________ September 8, 2009_ Technical Advisor (if Chair’s report) Date

WRAC ANNUAL PROGRESS REPORT – Year 2

PART II: Detail PROJECT TITLE: Coldwater disease prevention and control through vaccine

development and diagnostic improvements REPORT GIVEN IN YEAR: 2009 REPORTING PERIOD: September 1, 2008 – August 31, 2009 (2nd 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 (approved 10/08) Fourth Year Request: $81,639

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 Student: Amy Long University of Idaho Faculty participant: Devendra Shah Washington State University

PROJECT OBJECTIVES:

1. Identify potential vaccine candidates using in vivo-induced antigen technology. • 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

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 and a Postdoctoral Fellow (Rajesh Kumar) worked on this project from July 2008 to July 2009 at the University of Idaho (Cain) and Washington State University (Call), respectively. A workgroup meeting (conference call) was held in July 08 and another will be held in late September to discuss results to-date and upcoming needs for next year.

Objective 1: Identify potential vaccine candidates using in vivo-induced antigen technology. We previously identified a F. psychrophilum protein, ACP21, as a positive control that is putatively expressed in vivo. This determination was made by selecting the protein from previous in vivo expression work by our group (LaFrentz et al. In press) and then using a western blot assay that showed detection of a recombinant ACP21 protein with absorbed antisera while whole cell lysates from in vitro growth F. psychrophilum were negative (data not shown). The IVIAT library has already been constructed using a pET30 vector (3 frameshifts represented) in a BL21(DE3) E. coli host. Using absorbed antisera from convalescent rainbow trout, we were able to optimize the detection assay so that recombinant protein could be detected on a membrane after cultivation in 200 ul of broth culture within a 96-well plate (Fig. 1). Importantly, this was successful using only 10 ul of supernatant from the 200 ul culture. Concentration of the supernatant did not improve detection sensitivity (data not shown). We showed that this could be accomplished using either E. coli or by using Vibrio parahaemolyticus as an alternative expression host. Despite this success, we have been unable to migrate our protocol as illustrated in Figure 1 to screen IVIAT library plates owing primarily to excessive nonspecific background and insufficient analytic sensitivity combined with a high level of variance between each experiment (Fig. 2). This protocol also consumes a large volume of convalescent antisera. We have shown that robust detection can be achieved using 5 ml of cultured bacteria. Unfortunately, this is not practical for screening a full library. Our options for continuing along this line of work are limited. We can re-create the library using V. parahaemolyticus as the expression host as proposed previously, but other experiments in the lab suggest that we will encounter a similar degree of variance in detection and a high degree of background signal (data not shown). Consequently, it is uncertain if the library screen can be completed as originally proposed owing to a combination of limited sensitivity using absorbed antisera and insufficient protein expression using a 96-well plate format.

Despite our difficulties with the IVIAT library, we have developed a new resource for identification of proteins of interest in F. psychrophilum. Recent work by our group included development of an attenuated strain of F. psychrophilum. Attenuation was achieved by serial passage on rifampicin (antibiotic) plates, which led to identification of clone B.17 that has a high level of drug resistance without diminution of its ability to grow in broth culture. Rifampicin interferes with RNA polymerase activity and resistance is conferred by 1-2 point mutations in the beta subunit of the RNA polymerase holozenzyme. This method of serial passage has been used to develop attenuated strains of other pathogens including Flavobacterium columnare and Brucella abortus although it is worth noting that rifampicin resistance is no guarantee of attenuation as is abundantly clear from the existence of rifampicin resistant Mycobacterium tuberculosis. We have demonstrated that immunization of rainbow trout by injection or immersion with strain B.17 produces an IgM response and confers protection against injection challenge with F. psychrophilum (LaFrentz et al. 2008). We have conducted a preliminary investigation of strain B.17 to determine how it behaves differently from the parent strain CSF259.93. Specifically, we were interested in identifying unique proteins in the parent strain that were no longer expressed in the B.17 strain under the assumption that these proteins may be directly related to virulence. Using 2-dimensional PAGE experiments we have been able to identify a number of proteins that are unique to CSF259-93 (Fig. 3). Somewhat to our surprise, however, was the presence of a number of proteins that were unique to strain B.17. These findings provide two new avenues of investigation that we propose to pursue as an alternative to continuing work with the IVIAT library. First, we can identify the proteins that are differentially expressed using conventional mass spectrometry and preliminary work has provided the identity of several of these proteins. We will also determine which, if any, of the proteins in question are antigenic using Western blot analysis with convalescent trout antisera. Secondly, the dramatic change in protein expression profiles between CSF259-93 and B.17 could result from a number of factors including changes in the promoter binding characteristics of the RNA polymerase, or alteration with the interaction between the RNA polymerase and a key global transcriptional regulator, or alteration in the ability of strain B.17 to accurately “sense” its surroundings and respond accordingly. Regardless of the mechanism, it is most likely that strain B.17 is attenuated because it is “running the wrong program” rather than missing a single virulence factor. We submit that it is important to determine the mechanism by which transcriptional regulation is so dramatically altered because this effort could lead to a more direct and effective (limited chance for reversion) means to develop attenuated strains of bacteria. We have already identified a predicted point

mutation in the beta subunit DNA sequence from strain B.17. Our efforts will include determining if the differentially expressed proteins share a common promoter binding sequence and this work will be aided by the availability of two whole genome sequences including one for strain CSF259-93. Protein-capture and immunoprecipitation experiments will be combined with mass spectrometry to identify potential transcriptional regulators that bind the DNA sequences identify from the 2-D PAGE analysis. We will also replicate the serial passage experiments to determine if rifampicin resistance leads to similar outcome each time, both in terms of protein expression and virulence attenuation. We also wish to share with the committee some additional preliminary findings that support the value of our effort to identify unique proteins for immunization trials against F. psychrophilum. In a recent study we compared immunization of rainbow trout with a mixture of four antigenic proteins that were purified by conventional column chromatography versus the same four proteins that were administered as a mixture of crude lysates (Fig. 4). In both cases, the proteins were expressed using our V. parahaemolyticus expression system. From this challenge experiment it appears that (1) Vibrio lysate by itself has an adjuvant effect greater than Freund’s complete adjuvant (FCA). Secondly, immunization with a mixture of our four purified proteins produces no protective effect. If, however, the fish are immunized with a crude preparation of recombinant protein that includes lysate from V. parahaemolyticus, we see elevated protection relative to purified proteins and FCS alone (P<0.05). These findings show the potential for generating a protective immune response using crude lysates that are much cheaper to produce compared with purified subunit vaccines.This work also demonstrates the potential benefits of using V. parahaemolyticus lysate as an adjuvant. If this work is supported in subsequent experiments, it points to a more viable vaccine strategy and we may have identified a more effective expression system with adjuvant activity. Objective 2: Validate quantitative diagnostic assays (ELISA and ovarian fluid filtration FAT) ELISA Efforts to optimize the capture ELISA using the protocol initially developed by our lab were continued this year. It had been hypothesized that our antibody purification techniques were not

optimal thereby resulting in an antibody stock that had extraneous proteins which interfered with horseradish peroxidase (HRP) conjugation. Antibody purification protocols have been optimized and while extraneous proteins are still present to some degree, we are confident that they are not interfering with HRP conjugation. Purification of FL43 from ascites using the techniques that were used for the original development of the assay is not possible but, with our optimized protocols, the antibody concentration is actually higher (3 mg ml-1) than with the previous method. The technique for conjugating HRP to MAb FL43 was also refined. The original ELISA had two standard curves, one with PBS and one with kidney tissue from disease-free rainbow trout, both spiked with known concentrations of F. psychrophilum. We have attempted recreate both standard curves. Regardless of the different antibody purification techniques, the detection limit of the capture ELISA remained at 106 CFU ml-1. Since it appeared that increasing the sensitivity of the ELISA using HRP conjugated MAb FL43 would be difficult, it was decided to incorporate a biotin-streptavidin system into the assay to increase sensitivity. Avidin is a glycoprotein that has a high affinity for biotin so the two molecules form a stable, lasting bond. Amplification of the signal is a result of the four biotin binding sites on the avidin molecule which means it is able to bind more biotin per molecule. The addition of biotin and avidin does not change the specificity of the assay, it simply amplifies the pre-existing signal. For this assay, MAb FL43 was conjugated to biotin and used in place of MAb FL43 conjugated to HRP. The plate was then incubated with streptavidin conjugated to HRP. Once the biotin ELISA was optimized, we tested the detection limit of the ELISA with spiked kidney samples and found that it remains at approximately 106 CFU ml-1. Dr. Call recently suggested that the ELISA may actually have similar levels of sensitivity compared to the original but we are not seeing it because our method for determining the number of colony forming units per milliliter is different than that used when the ELISA was first developed. Typically, an exponentially growing broth culture of .F psychrophilum that has an optical density at 525 nm of 0.3 is used. This optical density corresponds to approximately 108 CFU ml-1 as determined by the 6 x 6 drop plate method. When the ELISA was first developed, the standard curve was done using a culture of F. psychrophilum that had an optical density at 540 nm of 0.6 which was thought to correspond to 108 CFU ml-1. However, it is possible the optical density reading did not correspond to 108 CFU ml-1 and this is why we are unable to reproduce the standard curve. Furthermore, F. psychrophilum is known to agglutinate when grown in broth, and even though broth cultures are vortexed before being serially diluted for plate counts and spiking kidney tissue, it is possible that early calculations did not represent the true number of bacteria present. Care is taken in current protocols to keep cultures from auto-agglutinating prior to plate counts. One way to confirm this is by running an ELISA using F. psychrophilum 259-93B.17. This strain was developed by our lab as an attenuated strain. Growth studies with this strain have shown that it does not agglutinate in broth culture as much as the parent strain. Both the parent strain and the B17 strain will be grown to an optical density of 0.6 at 540 nm, and the capture ELISA will be performed using the original method with MAb FL43 conjugated to HRP and not biotin. The assay will be further modified to include increasing antigen incubation time. Previously, the sample containing the antigen was incubated on the plate for two hours. We intend to try an overnight incubation to see if that increases the amount of binding to the capture antibody.

Challenge Experiment The purpose of this challenge experiment is to evaluate changes in ELISA optical density values over the course of a bacterial challenge. As the challenge is ongoing and the ELISA protocol is still being optimized, results are not included in this report. In brief, juvenile rainbow trout reared on pathogen-free municipal water and weighing approximately 5-10 g were split into three treatments: mock-infected, high dose, and low dose. The mock-infected was sterile PBS, the high dose was F. psychrophilum at a concentration of 107 CFU ml-1, and the low dose was 105 CFU ml-1. Each treatment was done in triplicate with 25 fish per tank for a total of 75 fish per treatment. Prior to injection, five fish were sampled for baseline readings. Kidney, liver, and spleen were streaked on TYES agar and kidney was extracted from each fish and pooled for ELISA. Imprints of kidney and spleen were also made for IFAT. On day 2 of the experiment, one fish was sampled from each tank. Again, kidney, liver and spleen were streaked, and spleen and kidney imprints were made. Kidneys from each treatment were pooled and homogenized. Random sampling has occurred every other day in this experiment. In addition, 20% of the daily mortalities are also being worked up using the same protocol. All kidney homogenates have been frozen at -80°C until they can be evaluated with ELISA. MF-FAT This past spring, ovarian fluid was aseptically collected from steelhead returning to Dworshak National Fish Hatchery (DNFH, Orofino, ID) and rainbow trout broodstock at the J. Perry Egan Fish Hatchery (JPEFH, Bricknell, UT). Samples were transported to the University of Idaho either on the day of collection (DNFH) or within 48 hours of collection (JPEFH). Once at UI, a sub-sample was removed for culture, and samples were then frozen at -80°C until needed. The sub-sample was plated on TYES agar and plates incubated at 15°C for 72 hours. After 72 hours, plates were examined for the presence of yellow-pigmented bacteria (YPB). Any YPB were re-streaked and frozen down for identification. Thirty-two samples from DNFH had YPB, and none of the samples from JPEFH had YPB. In fact, the plates from JPEFH were overgrown with white colonies after an incubation period of 48 hours. In all, we collected 122 samples from the two different hatcheries with the majority from DNFH. Of the 122 samples, 118 were analyzed by MF-FAT. For the MF-FAT, samples were processed according to the protocol previously developed by our lab. Fifty fields were counted per slide and samples were called positive if at least one F. psychrophilum cell was observed. Thirty-eight of the samples were positive for F. psychrophilum. JPEFH had the most positive samples, 47%, by MF-FAT even though none of the samples had YPB growth. This indicates the MF-FAT also works with samples that are not frozen immediately after collection and may be a more accurate tool for checking samples for F. psychrophilum than culture techniques. To determine the minimum number of CFU ml-1 that could be detected by the MF-FAT, ovarian fluid was aseptically collected from steelhead at DNFH, filtered through a 0.22 µm filter to remove any bacteria that may have been present, and stored at -80°C. An exponentially growing F. psychrophilum culture was serially diluted from 10-1 to 10-7 in the filtered ovarian fluid. MF-

FAT was done on all the dilutions as well as the undiluted culture. The number of CFU ml-1 in the bacterial culture was determined using the 6x6 drop plate method. As before, 50 fields were counted per filter. Cell counts were plotted against the number of CFU ml-1 and a linear regression was done (Fig. 5).

Figure 5. Total # of F. psychrophilum cells as determined by MF-FAT versus CFU ml-1. The standard rule of thumb for direct cell counts is that a minimum of 150 cells should be counted to ensure counts are statistically significant. Using the equation of the line and the 150 cell limit, we were able to determine that the MF-FAT is able to detect as few as 8.8 x 103 CFU ml-1. Considering that we determined CFU ml-1 using the 6x6 drop plate method which may be underestimating the number of F. psychrophilum cells, there is a chance that our detection limit is actually lower than 103 CFU ml-1. This will be re-evaluated once we have developed an accurate and repeatable method for determining CFU ml-1. We will also determine the minimum number of cells per milliliter of sample that can be detected using the MF-FAT. IFAT The indirect FAT protocol was worked out this past year as a way to rapidly test samples for F. psychrophilum. For the IFAT, MAb FL43 is conjugated to Alexa Fluor® 488 using the same methods for the MF-FAT. The IFAT can be done within two hours using either tissue imprints, specifically spleen and kidney, or whole fish homogenate. We have been able to detect bacteria in using this method in samples from rainbow trout that were directly challenged with F. psychrophilum. However, tissue imprints have been done for both F. psychrophilum and mock-

infected fish from the current challenge experiment. The slides are labeled in a manner that does not indicate the status of the fish and so we can apply a blind test to determine if the IFAT is able to discriminate between uninfected and infected fish without being biased as to the disease status of the fish. Objective 3: Based on results from objective 2: Develop other assays (e.g. real-time quantitative PCR) for quantification of infection in ovarian fluid. No progress to date. Objective 4: Develop an integrated outreach program to meet stakeholder needs. Outreach activities have resulted in of an article describing this project to WRAC that was published in Waterlines [Vol 15(1)].

USEFULNESS OF FINDINGS: Findings thus far, along with previous development of the monoclonal antibody (FL43) has led to a license agreement between the University of Idaho and an antibody company (Immunoprecise) out of Vancouver, BC. This company has now produced FL43 and it is available commercially to diagnostic laboratories, research labs, and others wishing to test for CWD. Based on further results from this WRAC project, Immunoprecise will develop ELISA and FAT kits for the industry and research labs. WORK PLANNED FOR NEXT YEAR: Objective 1: Identify potential vaccine candidates using in vivo-induced antigen technology. We can take two different courses of action in the next year. If the committee prefers, we will continue work with the IVIAT library based on a Vibrio parahaemolyticus expression host instead of the E. coli expression host. To be successful will require generating more convalescent antisera, which can be achieved using facilities at the Cain lab or at Clear Springs. Regenerating the library requires cloning and transformation of V. parahaemolyticus followed by arraying clones into 96-well plates as we have done before. There are no technical obstacles to this procedure. There remains the question of whether or not we will gain sufficient analytic sensitivity from V. parahaemolyticus expression (increased protein yield) in a 96-well format to allow us to overcome the requirement for a 1:50 dilution of absorbed antisera. The latter is most likely responsible for the high level of background apparent from our dot blots (see Fig. 2 above). Preliminary tests in the Call lab indicate that we can probably expect to continue having trouble with a limited signal to noise ratio. Our second course of action is to re-direct our efforts to a more complete characterization of the B.17 strain. This would involve two lines of investigation: o Examine differentially expressed proteins in CSF259-93. This work would identify

additional candidate genes that are putatively involved in virulence and determine if these proteins can be used as vaccine candidates. This work would follow our previous methods and successful efforts to characterize differentially expressed proteins between CSF259-93

and the non-virulent ATCC 49418 strain (Sudheesh et al. 2007), as well as our successful efforts to identify differentially expressed proteins by CSF259-93 after in vivo growth (compared with in vitro growth) and in response to iron limiting and non-limiting conditions (LaFrentz et al., In press).

o Identify the mechanism by which strain B.17 has been attenuated. Our working hypothesis is that attenuation has resulted from altered global transcriptional regulation and that this pattern of change is repeatable, predictive of attenuation, and can be engineered independently from rifampicin passage. To test this hypothesis and the alternative mechanisms (expression differences due solely to changes in RNA polymerase activity or due to changes in environmental sensing pathways) we will complete our proteomic and sequence analysis to identify transcriptional binding sites that we will then use as “bait” to capture and identify transcriptional regulators involved in this process. We will use parallel rifampicin passage experiments to determine if the same pattern of transcriptional alterations is a repeatable phenomenon. Proteomic and sequence analysis will be used to confirm changes in the passaged strains and we will determine if strains with similar characteristics to 259-93B.17 are attenuated. Finally, we will generate deletion mutants to confirm their role in altered transcriptional regulation and attenuation. The rationale for this work is that we will know the precise mechanism involved in attenuation, which means that we will have the opportunity to design a more robust attenuated strain, but more importantly, we will identify a predictable means for engineering attenuated strains with the clear implications that this procedure can be used to create attenuated strains of other important bacterial pathogens.

General methods. We have published experience with the methods needed to conduct these studies including analysis of 2-D PAGE experiments (Sudheesh et al. 2007; Plant et al. 2009; LaFrentz et al. in press); production of recombinant proteins from AT-rich coding sequences found in F. psychrophilum (Shah et al. 2008); experience with sequence analysis; experience with analysis of transcriptional regulators (Zhou et al. 2008); and experience with challenge models needed to test for attenuation LaFrentz et al. 2002, 2003, 2004, 2008). Consequently, the details provided below illustrate our experimental design with additional methodological details if needed to clarify our strategy. We will focus our analysis CSF259-93 and B.17. Use 2D-PAGE comparisons to identify proteomic differences associated with attenuation. Primary comparison and positive identification of proteins unique to either the attenuated rifampicin-resistant strains or the parent strain of F. psychrophilum will follow established procedures (Sudheesh et al. 2007). Differentially expressed spots, as seen in Fig. 3 above, will be identified by gel comparison and excised. Following excision, gel spots will be placed in 5% acetic acid and submitted to the Proteomics Core Facility at Washington State University for LC-MS/MS analysis. The peak lists obtained for each spot will be searched against the F. psychrophilum (ATCC 49511) genome (EMBL database, accession number AM398681) and CSF259-93 genome (G. Wiens, unpub. data) using the Mascot searching algorithm (Matrix Science, London, UK). We expect to identify 6-10 differentially produced proteins each from CSF 259-93 and 259-93B.17. Proteins unique to the CSF259-93 strain are potential targets for the study of molecular pathogenesis of coldwater disease, and possible production of attenuated deletion mutants. Non-coding 5-prime regions of identified genes will be analyzed for transcriptional binding motifs where we expect genes that are uniquely expressed in one strain

versus the other will have different binding motifs that are indicative of interaction with different transcriptional regulators. We will also determine the DNA sequence for RNA polymerase subunit genes and nine putative transcriptional regulators or anti-sigma factors (n=2) that are annotated in the F. psychrophilum genome. All of these sequences are available from the whole genome sequence for primer design and sequencing will include flanking sequences. Identify transcriptional regulators. We will use a “bait-and-capture” strategy to determine which transcriptional regulator proteins are binding the genes that are unique to CSF259-93 and B.17. Double-stranded oligonucleotides (“bait”) will be assembled for conserved DNA binding motifs identified from the proteomic analysis (Liu et al. 2004). If no common motifs are identified, we will include all 5-prime binding regions for the identified proteins individually or as a mixture. Double-stranded oligonucleotides will be up to 60 bp in length with the putative DNA binding region centrally located. One strand will be biotinylated. After annealing complementary strands, the oligonucleotides (“bait”) will be mixed with cell lysate where DNA and proteins can interact. Streptavidin-coated paramagnetic beads will then be added and after binding to biotinylated DNA and the magnetic beads will be retained and washed. Proteins that are bound to the oligonucleotides will be separated by TCA precipitation, solubilization and detection by SDS PAGE. Protein bands detected in the gel will be isolated and analyzed by LC-MS/MS analysis to identify the proteins as described above. If paramagnetic beads do not provide sufficient yield for analysis, we will use an immunoprecipitation technique to retrieve protein-DNA complexes by capturing bait:target complexes through either an antibody against biotin or a streptavidin moiety (Grainger and Busby 2008). We expect to identify two transcriptional regulator proteins—one that is unique to CSF259-93 and the other that is unique to B.17 for the culture conditions used in this study. Controls will include reciprocal experiments where the B.17 bait and CSF259-93 baits are switched. RT-PCR experiments will be used to determine if the transcriptional regulators are differentially expressed between the five strains of interest. We will subsequently determine if there are differences in the copy number for transcriptional regulator mRNA. If global expression changes are due to altered expression of the transcriptional regulators (e.g., due to direct mutations or due to altered two-component signaling) then we expect to detect differences in mRNA copy number between CSF259-93 and B17. If altered transcriptional patterns are due to changes in the interaction between RNA polymerase and transcriptional regulators, then mRNA copy numbers may not differ and this would also result in detection of reciprocal transcriptional regulators for the bait experiments described above. RT-PCR experiments will be designed for detection by SYBR green staining and will employ a ΔCP method with 16S rRNA sequence as the normalizing target for relative quantification Pfaffl 2001). Repeatability of the attenuation mechanism. It is not known if attenuation by passage on rifampicin plates results from a limited number of genomic alterations or whether this is largely a random process with a multitude of outcomes. The objective of this experiment is to determine if the observed transcriptional changes in the attenuated strain are a predictable outcome. The working hypothesis is that passage of F. psychrophilum on rifampicin plates will produce multiple transcriptional profiles, but any profile consistent with strain 259-93B.17 will be attenuated. The experimental procedures will follow LaFrentz et al. (2008) and passaged strains will be banked every generation for retrospective analysis of transcriptional changes. Passage experiments will be conducted for strain CSF259-93 and for strain THC02/90 with five clones of

each strain passaged in parallel. Strain THC02/90 will be included in this study because we are able to genetically manipulate this strain (data not shown) and because we have demonstrated that this strain is virulent in our challenge model (data not shown). No one has successfully manipulated strain CSF259-93, presumably due to the presence of restriction modification systems (Alvarez et al. 2004; Soule et al. 2005). We expect that by the time passage achieves resistance to >250 ug/ml rifampicin we will observe strains that have a very similar proteomic profile as B.17. This will be determined by 2-D gel electrophoresis and sequence analysis focused on markers identified in the sequencing experiments described above. Attenuation will be tested using an in vivo challenge model following the methods of LaFrentz et al. (2008). Attenuation by gene deletion. The experiments described in this section will only be pursued provided sufficient time and resources remain in the project period. If our hypothesis is correct and strain B.17 is attenuated due to alteration in transcriptional regulation, then we expect that deletion of the key transcriptional regulator will not be lethal, but this deletion will recapitulate the altered transcriptional program. Both Alvarez et al.(2004) and our lab (data not shown) have successfully mutagenized strain THC02/90 using plasmid pEP4351, which encodes transposon Tn4351. Briefly, E. coli BW19851 is used to transfer pEP4351 into F. psychrophilum by mixing cultures (ca. 108

cells each) and plating on EAOS medium. After 48 h incubation at 20°C, during which time conjugation occurs, cells are scraped-off the plates and grown on selective media for 4 to 7 days. Counter selection against E. coli is not needed because the antibiotic resistance genes encoded by pEP4351 are not expressed in E. coli. Plasmid pEP4351 is a suicide vector (no origin of replication compatible with F. psychrophilum) so only strains with successful chromosomal integration of Tn4351 are detected. Alvarez et al. (2008) screened a similar library based on phenotypic tests, but this option will not be available for high throughput screening unless we identify a suitable phenotype in our earlier studies. Consequently, we will transfer mutated strains to 96-well plates and screen by PCR. To make this process as efficient as possible, we will isolate 17,200 transposon mutated clones into 96-well plates, which will provide 95% probability that we have collected all nonlethal mutations assuming the genome is 2.86 MB in size and average size of a coding region is 1,000 bp. This will be accomplished using a robotic picker as we have recently done to screen a 136,000 clone library from another study (Call et al. unpub. data). Clones will then be recombined in 96-well plates so that 10 strains are represented in each well. PCR primers will be designed spanning the gene of interest and this assay will be used to screen 18 plates by PCR. Positive mutants will be detected by the presence of a larger than expected band as detected by agarose electrophoresis. Strains composing a positive well will be rescreened as individual strains to identify the clone of interest. PCR and sequencing will be used to confirm Tn4351 insertion and 2-D PAGE will be used to determine if the pattern of global transcription changes as expected. If successful, we will generate a complemented strain by inserting the wild-type gene in pCP23 for expression in F. psychrophilum (Alvarez et al. 2004). Expected Outcomes. We expect to identify one or more mutations in strain 259-93B.17 that produce changes in global transcriptional regulation of the F. psychrophilum genome. We also expect to demonstrate that these changes are repeatable, predictable, and inducible by gene deletion. These findings will be significant because they will, for the first time, provide a mechanistic understanding of how attenuation occurs in rifampicin passaged Flavobacterium. Of greater importance, however, is that these findings are likely to be generalizable to other

important bacterial pathogens and our results will provide invaluable guidance for generating predictable attenuation without the need for reliance on passage experiments. Anticipated Problems. We have experience with all of the methods described herein and we do not anticipate any technical problems with this series of experiments. It is possible that attenuation is not a predictable outcome, but even if this is the case, if we can mechanistically replicate the attenuation observed for strain B.17, we will provide one model for how a pathogen can be attenuated for vaccine preparations. It is also possible that our passage experiments with THC02/90 will not recapitulate the proteomic changes seen in B.17. In this case we will determine which if any of the rifampicin passaged THC02/90 strains are attenuated. Mutation of a key transcriptional regulator in THC02/90 might produce a protein pattern that is distinct from B.17 in part because there may be a number of other genetic differences between these strains as they represent distinct genetic lineages of F. psychrophilum. Nevertheless, if the protein pattern from the passaged strains is consistent with the deletion mutant and this also produces an attenuated strain, our hypothesis will be supported. Objective 2: Validate quantitative diagnostic assays (ELISA and ovarian fluid filtration FAT) It is possible that we may have to accept the ELISA will not be able to detect as low as 103 CFU ml-1. However, in reviewing previous protocols we feel strongly that initial reports of sensitivity may not have been completely accurate due to methodology employed. It is known that CSF 259-93 auto-agglutinates, and this characteristic would result in lower CFU ml-1 counts due to clumps of bacteria settling down on agar plates. It is hypothesized that earlier estimates may have reflected lower bacterial numbers than were actually present. We are attempting to confirm this, but currently are taking great care to disrupt and distribute bacteria evenly in solution prior to quantification to accurately reflect the numbers of viable bacteria. Our goal is still to lower the detection limit to approximately 104 CFU ml-1. However, we will be testing recently collected field samples from the Lower Elwha hatchery to see if results are comparable with previous findings (Lindstrom et al. 2009). In addition, our current fish challenges will provide us with controlled samples to track infection rates over time. Based on this, we plan to begin the first round of sampling at Troutlodge by the end of fall semester or early next year. Briefly, samples of kidney and ovarian fluid from up to 60 female broodstock will be collected at various times during the year. Samples will come from replacement broodstock (3 year olds) at Troutlodge in order to obtain both kidney and ovarian samples because these fish are normally culled from the population at that time. Fertilized eggs from these fish will be kept in isolated incubators available at Troutlodge and kidney/ovarian samples will be sent to University of Idaho for analysis. During the incubation period, results from ELISA (kidney tissues), FAT (ovarian tissues) and culture (kidney and ovarian fluid) will be obtained as a means of selecting appropriate groups. Eggs from up to 10 individuals that have either extremely high or extremely low levels of infection will be shipped to UI when they reach the eyed stage. As we are attempting to assess how broodstock infection levels at time of spawning relate to outbreaks in progeny, it may be easier to do this if we use extremes in infection levels for the first round of experiments. This will also make determining correlation easier.

Progeny will be reared in the wetlab facilities at UI. Progeny will be separated based on the infection level in the parents. Once progeny reach 0.5 g, fish will be subdivided and stocked into triplicate in 20 l tanks (50 fish/tank). Fish will be subjected to chronic stress conditions and monitored for disease outbreaks. It is possible that the bacteria may be present in fish without causing any external symptoms, and as such, fish will be randomly sampled throughout the experiment. Kidney, liver, and spleen will be streaked on TYES agar plates and monitored for yellow-pigmented bacteria growth. All mortalities will be examined to determine cause of death. Clinical symptoms consistent with CWD and isolation of F. psychophilum in the absence of other known pathogens will provide strong evidence from vertical transmission. All progeny will be assayed for F. psychrophilum prior to stress trials and progeny from broodstock testing negative for F. psychrophilum will be included in all trials and serve as a negative control. An additional method for confirming vertical transmission is comparing the genetic fingerprint of YPB isolated from progeny to those isolated from broodstock using pulsed-field gel electrophoresis. Protocols for this technique have been worked out by Dr. Call's lab (Chen et al. 2008) and so we should be able to obtain genetic fingerprints of the different isolates quickly. It should be noted that the ability to mimic production conditions in the lab or detect F. psychrophilum in progeny from the above trials may be difficult, and CWD outbreaks may not occur. Therefore, we will also challenge groups using our standardized procedures to determine susceptibility differences in relation to broodstock infection levels. Although precedence for such a relationship is unknown, it is possible that additional factors beyond vertical transmission may play into disease outbreaks and broodstock infection levels. We have speculated that because F. psychrophilum can be found in many water supplies, progeny may show varied levels of susceptibility. Since our experimental design will allow this to be tested easily, it has been included as a component of this project. It is well established that different strains of fish show varied degrees of susceptibility to a range of pathogens. In fact, selection programs for trout have been implemented at USDA (Cold and Coolwater Laboratory) and Clear Springs Foods, Inc. for both disease resistance and growth. It is possible that this susceptibility of progeny relates to the carrier status of broodstock. Therefore, by challenging fish from each group we may observe higher mortality from progeny of heavily infected broodstock (even in the absence of measurable vertical transmission). If this were indeed the case, then it would further support the culling of infected broodstock/eggs. To determine a threshold for culling, all mortalities that occur in the trials will be compared using ELISA OD values and FAT cell counts/ml of ovarian fluid. This information will be used to establish “cut-off” levels for each assay for use in making decisions associated with broodstock or egg culling. Objective 3: Based on results from objective 2: Develop other assays (e.g. real-time quantitative PCR) for quantification of infection in ovarian fluid. Development of a real-time quantitative PCR assay for detecting low levels of F. psychrophilum in ovarian fluid as well as kidney tissue will be done this year. Ideally, the probe for this assay will be specific to the gene that encodes the antigen that is recognized by MAb FL43. The sequence of this gene is currently unknown; however, Dr. Call's lab is close to elucidating the sequence. Once that sequence is known and assuming that is it is specific to F. psychrophilum

and does not share homology with any other genes, we will begin developing protocols for extracting bacterial DNA from samples and optimizing the assay. If we are not able to use the sequence for the outer membrane protein, the genome of F. psychrophilum has been sequenced and can be mined for potential targets for this assay. Once the target has been decided upon, the next step will be to optimize running conditions for the real-time qPCR assay. In addition to optimizing running conditions, we will also have to ensure that our assay is specific to F. psychrophilum. We can do so by running the assay with the 67 different strains of F. psychrophilum which are maintained by our lab as well as with different Flavobacterium species and different fish pathogens. The detection limit of the assay will also have to be determined for both kidney tissue and ovarian fluid. To do so, kidney tissues and ovarian fluid will be spiked with serially diluted F. psychrophilum, bacterial DNA extracted from the samples, and DNA used as the template for the real-time qPCR assay. The real-time qPCR assay will then be tested on samples that we have already tested for F psychrophilum by other methods such as ELISA and MF-FAT. Ovarian fluid and kidney tissue sampled from broodstock at Troutlodge and used for Objective 2 will be preserved at -80°C for this purpose. Additionally, ovarian fluid and kidney tissue samples have been collected from Lower Elwha hatchery and Dworshak National Fish Hatchery and can be used for this purpose. Objective 4: Develop an integrated outreach program to meet stakeholder needs. Increase awareness of project and research progress through popular press articles in aquaculture newsletters and presentations at aquaculture meetings. The integrated approach will follow established guidelines in relation to each objective (see original proposal).

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, McBride MJ, Guijarro JA. Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum. Applied Environmental Microbiology. 2004 70:581-7 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. 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 (In Press)

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 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 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 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: Currently, the primary impact is commercial availability of monoclonal antibody FL43. The agreement with Immunoprecise and their involvement will be pivotal in implementing findings from the diagnostic component of this research. SUPPORT:

Year WRAC-USDA Funding University Industry Other

Federal Other Total Total Support

2008 $81,555.00 $81,555.00 2009 $80,043.00 $80,043.00

2010 $81,637.00 (approved 10/08) $81,637.00

Total $81,555.00 $243,235.00 PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED: Refereed publications: 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 (In Press) 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 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 General articles: Cain, K.D. 2009. Strategies for Control and Prevention of Coldwater Disease. Waterlines newsletter 15 (1): p. 18-20 Presentations: 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.

SUBMITTED BY:______________________________________September 8, 2009____ Title: (Work Group Chair or PI) Date

APPROVED: ______ ________September 8, 2009________ Technical Advisor (if Chair’s report) Date