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WRAC TERMINATION REPORT PART I: Summary PROJECT TITLE: COLDWATER DISEASE PREVENTION AND CONTROL THROUGH VACCINE DEVELOPMENT AND DIAGNOSTIC IMPROVEMENTS REPORT GIVEN IN YEAR: 2011 PROJECT WORK PERIOD: 5/1/2008-present; no-cost extension approved through 9/30/12) AUTHORS: Ken Cain and Douglas Call PARTICIPANTS: Kenneth Cain* (Work Group Chair) Univ. of Idaho Douglas Call* Wash. State Univ. Scott LaPatra Clear Springs Foods Gary Fornshell* (Outreach Coordinator) University of Idaho Greg Weins USDA, West Virginia Technical Advisor: Gael Kurath USGS, Washington Industry Advisor: Jim Parsons Troutlodge, Wash. Graduate Students: Amy Long/Karol Gliniewicz UI/WSU Faculty participant: Devendra Shah Wash. State Univ. REASON for TERMINATION: End of 4 year project, funds terminated 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. This has included developing and validating improved diagnostic assays and exploring vaccine development by identifying possible bacterial gene targets and expanding work on an existing attenuated vaccine. 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. This objective was modified to include studies of both subunit vaccines and the mechanism responsible for attenuation of strain CSF259.93.B17. 2. Validate quantitative diagnostic assays and assess utility for assessing the risk of vertical transmission. 3. Develop alternative assays for quantification of infection in ovarian fluid. 4. Develop an integrated outreach program to meet stakeholder needs. 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

Transcript of University of Washington - WRAC TERMINATION REPORT PROJECT … · 2019. 6. 21. · PROJECT...

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WRAC TERMINATION REPORT

PART I: Summary

PROJECT TITLE: COLDWATER DISEASE PREVENTION AND

CONTROL THROUGH VACCINE DEVELOPMENT

AND DIAGNOSTIC IMPROVEMENTS

REPORT GIVEN IN YEAR: 2011

PROJECT WORK PERIOD: 5/1/2008-present; no-cost extension approved through 9/30/12)

AUTHORS: Ken Cain and Douglas Call

PARTICIPANTS: Kenneth Cain* (Work Group Chair) Univ. of Idaho

Douglas Call* Wash. State Univ.

Scott LaPatra Clear Springs Foods

Gary Fornshell* (Outreach Coordinator) University of Idaho

Greg Weins USDA, West Virginia

Technical Advisor: Gael Kurath USGS, Washington

Industry Advisor: Jim Parsons Troutlodge, Wash.

Graduate Students: Amy Long/Karol Gliniewicz UI/WSU

Faculty participant: Devendra Shah Wash. State Univ.

REASON for TERMINATION: End of 4 year project, funds terminated

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.

This has included developing and validating improved diagnostic assays and exploring vaccine

development by identifying possible bacterial gene targets and expanding work on an existing

attenuated vaccine. 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. This objective was modified to include studies of both subunit vaccines and the

mechanism responsible for attenuation of strain CSF259.93.B17.

2. Validate quantitative diagnostic assays and assess utility for assessing the risk of

vertical transmission.

3. Develop alternative assays for quantification of infection in ovarian fluid.

4. Develop an integrated outreach program to meet stakeholder needs.

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

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Kumar) worked on this project from July 2008 to July 2009, and a PhD student (Karol

Gliniewicz) joined Dr. Call’s lab in 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. This termination report provides a summary of results

to date, but work is continuing through next year to effectively complete ongoing research and

prepare outreach materials

Objective 1: This objective was modified to include studies of both subunit vaccines and the

mechanism responsible for attenuation of strain CSF259.93.B17. An attenuated strain of F.

psychrophilum (CSF259.93.B17) was originally produced by serial passage on agar plates that

contain increasing concentrations of an antibiotic called rifampicin. Resistance to this drug is

normally conferred by point mutations in the rpoB gene, which produces a subunit of the RNA

polymerase holoenzyme. We verified that CSF259.93.B17 has an expected mutation in this gene.

We have also completed the comparative proteomic analysis for CSF259.93 (wild-type) and

attenuated strain for which we identified eight and six proteins that were uniquely up-regulated

in the wild-type and attenuated strains, respectively. Using a western blot procedure with our 2-

dimensional gels and mass spectrophotometry we also identified the antigen that is targeted by

our diagnostic antibody, FL-43 (see below). We completed a subunit vaccine trial using

recombinant FP1493 followed by challenge using CSF259.93, but detected no evidence for a

protective immune response despite elevated serum titer against FP1493. A paper describing this

work has been submitted for peer-review.

Our working hypothesis is that CSF259.93.B17 is attenuated because the mutation in the rpoB

gene interferes with the ability of the RNA polymerase holoenzyme to bind to promoter

sequences or it interferes with the polymerase interaction with sigma factors. To test this

hypothesis we are in the process of generating a “knock-in” mutant. If proteomic and attenuation

changes are recapitulated by the knock-in experiment, this will support the hypothesis (in

progress). In the past year we conducted a 454 sequencing experiment of the wild-type and

attenuated strains. This experiment (which was completed at no additional charge to the project)

identified 24 nonsynonymous mutations in the genome of the attenuated strain (there were only 8

for the wild-type strain). This high degree of mutation raises the possibility that attenuation

results from mutations in genes other than rpoB, although we cannot ascertain if the mutation

process was due to long-term selection on agar plates or due to the influence of rifampicin

selection pressure. To examine this alternative further we have generated new passaged strains

with and without rifampicin for a second genome analysis (in progress).

With the identity of FP1493 known and the fact that we know that its expression is influenced by

iron availability, we determined if growing the B.17 strain in iron limited media would improve

the competency for inducing a protective immune response. Coho salmon were therefore

vaccinated with either B.17 grown in TYES or in TYES with an iron chelator (2,2-bipyridyl;

DPD). Injection and immersion vaccination strategies were tested in this trial. The live

attenuated vaccine protected Coho salmon against a virulent strain of F. psychrophilum in both

the immersion and injection trials. Antibody titers were significantly higher in immunized fish

versus non-immunized fish at 4, 6, and 12 weeks post-vaccination. Overall, statistically

significant protection for immersion immunized fish was only observed in groups that were

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vaccinated with B.17 grown under iron-limited conditions consistent with improved competency

of the vaccine strain.

Objective 2: Validate quantitative diagnostic assays and assess utility for assessing the risk

of vertical transmission. Both the ELISA and MF-FAT have been validated as diagnostic

assays (Long et al., in review). In addition, bacteriological culture and a commonly used nested

PCR protocol (Taylor 2004) were also validated using a wide range of field samples. The ELISA

has the highest diagnostic sensitivity (0.97) and specificity (0.98). The sensitivity and specificity

of the ovarian fluid MF-FAT were both low.

Two separate trials were used to examine the link between broodstock infection levels and risk

of BCWD outbreaks in progeny; one trial with rainbow trout and the other with Coho salmon.

Using our diagnostic assays we selected five families for each trial that had varying levels of

infection. Eyed eggs from each family were shipped to the University of Idaho (UI) and progeny

were sampled for F. psychrophilum upon arrival and then on a regular basis for the next two

months in both trials. F. psychrophilum was detected within eggs upon arrival at UI and after

disinfection, indicating that vertical transmission of the bacterium had occurred. Once fish

reached an appropriate mass, stress experiments were initiated in an attempt to induce a BCWD

outbreak in progeny and relate this to broodstock infection level. We also conducted a

susceptibility trial using the Coho and found some evidence for differences between families..

While the link between broodstock infection levels and risk of progeny outbreaks is still

unknown, the ELISA can be used to assess antigen levels in broodstock and progeny. The

ELISA and other diagnostic assays including nested PCR and quantitative PCR can be used as

part of a health management plan to decrease the overall frequency of F. psychrophilum infected

fish at a facility. Additional work is underway to evaluate broodstock samples from six

hatcheries as part of an effort to determine prevalence of F. psychrophilum in spawning

populations. Early analysis indicates that prevalence is generally higher in broodstock from

hatcheries where fish are returning to spawn than those from commercial rearing facilities.

Objective 3: Develop alternative assays for quantification of infection in ovarian fluid.

Development of a quantitative PCR assay for ovarian fluid is underway. The target gene for the

assay putatively encodes the outer membrane protein (FP1493) that is the target of MAb FL43.

While we have been able to develop the assay for pure bacterial cultures and detect the gene,

extraction of bacterial DNA from ovarian fluid has proven difficult. We are currently optimizing

the extraction technique and anticipate completing this in the next two to three months. Attempts

to optimize the ELISA for ovarian fluid were unsuccessful because of a consistently poor signal

to noise ratio.

An extension of this work is continuing in partnership with a company “Infoscitex” a USDA

SBIR Phase I grant subaward. The work includes the development of a qPCR assay that uses

aptamers designed to the outer membrane of F. psychrophilum. Four targets have identified and

isolated which was the goal of Phase I. If Phase II is funded, we will begin work on detecting the

targets in ovarian fluid as well as tissue samples.

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Objective 4: Develop an integrated outreach program to meet stakeholder needs. Outreach

activities have resulted in two articles in Waterlines describing this WRAC project and

additional information highlight in the fall issue of Trout Talk. There have been media releases

and reports on our vaccine work along with numerous presentations at professional and other

meetings. In summer 2011, we participated in the University of Idaho's Center for Research on

Invasive Species and Small Populations (CRISSP) Research Experience for Undergraduates

(REU) program (and NSF funded program). An undergraduate from Eckerd College was

selected to work on the CSF 259-93 B.17 vaccine efficacy in Coho salmon project. In addition to

gaining valuable lab experience, the undergraduate student also presented the results of her

project to her peers at the conclusion of the program. Additionally, in July 2011, we led a

workshop at the Salmon Disease Course at Oregon State University. During the day-long

workshop, participants carried out an ELISA with MAb FL43 using kidney from fish injected

with F. psychrophilum. Participants in the course represented state, provincial, tribal, and federal

agencies as well as industry including Schering-Plough and Marine Harvest. Additional WRAC

publications will be developed following completion of current studies.

IMPACTS: There is a strong need for public and private aquaculture facilities to have additional

control and management options for CWD. One deliverable from this project 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. Diagnostic assays to cull infected broodstock are established and

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 and they are available as downloadable pdfs directly from their

website. The other deliverable will hopefully be a commercialized vaccine for CWD. The B.17

vaccine was patented by the University of Idaho in June 2010 and is currently being field tested

for efficacy. The UI press release about the vaccine was sent to a broad array of stakeholders

including Idaho trout growers. Recent experiments showing enhanced protection of the B.17

vaccine are viewed as potential enabling technology and a provisional patent application was

filed in August, 2011 to protect improved methodology.

RECOMMENDED FOLLOW-UP ACTIVITIES: We will complete the experiments outlined

above to determine the mechanism that is responsible for attenuation of CSF259.93.B17.

Ongoing work will continue through this next year to complete development of qPCR assays and

validate for use on ovarian fluid. We will complete evaluation of the mechanisms associated

with attenuation and wrap up work aimed at relating broodstock infection levels to risk of

disease in progeny.

Outreach activities will be a primary focus over the next year and beyond. Currently, the

patented vaccine is under field evaluation through a partnership with Aquatic Life Sciences who

has signed an option agreement with UI to licensing the patent. If results are promising, it is

expected that the vaccine could be commercialized and sold under a USDA conditional license

approval as early as January 2012. If commercialization of this vaccine occurs it may then be

possible to determine long term impact due to adoption and implementation of a vaccine to

control CWD and subsequent reduction of mortalities due to CWD. A survey of the target

audience or aquaculture vaccine manufacturers may provide the information needed for long

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term impact evaluation. The results of the initial vaccine field trial will be presented to the

Pacific Northwest Fish Health Protection Committee annual meeting and the joint US Trout

Farmers Association and Idaho Aquaculture Association Fall Conference in September 2011. If

the vaccine is effective, a downloadable WRAC outreach publication that will briefly cover

(there’s already a WRAC CWD publication) CWD, what it is, how to diagnosis it and its impact

and then in great detail how to use the vaccine and expected results based on the

immunization/challenge and field results. In addition, a section will be added to the WRAC

outreach publication describing the methodology of the diagnostic tools, how to apply the tools

for rapid CWD diagnosis and/or implementation of a broodstock and/or egg culling program.

The results would provide effective early detection of disease and treatment of juvenile fish and

possible long term reduction of disease if culling programs are implemented. The group will

follow-up with ImmunoPrecise and fish health labs to quantify impacts through the sale and use

of the monoclonal antibody FL43. One to two years after project completion a survey of the

target audience will attempt to determine the extent of diagnostic tool use and if broodstock

and/or egg culling programs are used to minimize CWD outbreaks. If the vaccine is

commercialized then workshops for private and public salmonid hatchery personnel will be held

to explain and demonstrate how to use the vaccine and incorporate the diagnostic tool for early

detection of the disease. An impact statement will be written after an evaluation of the

deliverables to industry and other stakeholders.

SUPPORT:

Year

WRAC-

USDA

Funding

University Industry Other

Federal Other Total

Total

Support

2008 $81,555 $81,555

2009 $80,043 $80,043

2010 $81,637 $81,637

2011 $81,639 $81,639

Total $324,874 $324,874

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PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED:

Refereed publications:

Plant, KP, SE LaPatra, DR Call, and Cain, KD. Immunization of rainbow trout (Oncorhynchus

mykiss) with Flavobacterium psychrophilum gliding motility protein N. Journal of Fish

Diseases (In review)

Long, A, MP Polinski, DR Call, and KD Cain. Validation of diagnostic assays to screen

broodstock for Flavobacterium psychrophilum infections. Journal of Fish Diseases (In review)

Gliniewicz, K, KP Plant, SE LaPatra, BR LaFrentz, K Cain, KR Snekvik and DR Call.

Comparative proteomic analysis of virulent and rifampicin attenuated Flavobacterium

psychrophilum. Journal of Fish Diseases (In review)

LaFrentz, B.R., LaPatra, S.E., Call, D.R., Wiens, G.D., and Cain, K.D. 2011. Identification of

Immunogenic proteins within distinct molecular mass fractions of Flavobacterium

psychrophilum. Journal of Fish Diseases (In Press)

Plant, KP, SE LaPatra, DR Call, and KD Cain. 2011. Immunization of rainbow trout

(Oncorhynchus mykiss) with Flavobacterium psychrophilum proteins elongation factor-Tu, SufB

Fe-S assembly protein and ATP synthaseβ. Journal of Fish Diseases 34, 247-250

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

General articles:

Cain, KD. 2009. Strategies for Control and Prevention of Coldwater Disease. Waterlines

newsletter 15 (1): p. 18-20.

Cain, KD and DR Call. 2010. Coldwater disease. Waterlines Newsletter, Spring 2010, p10.

Cain, K and DR Call. Coldwater Disease Research. Trout Talk, Fall, 2011.

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Presentations:

Cain et al. A potential vaccine to control bacterial coldwater disease. US Trout Farmers

Association and Idaho Aquaculture Association Fall Conference. Twin Falls, ID. Sept. 29-Oct.

1, 2011.

Cain and Zinn. BCWD Vaccine Development. 56th Pacific Northwest Fish Health Protection

Committee Annual Meeting, Portland, OR. Sept. 21-22, 2011.

Cain, K.D. Research overview and update. University of Tasmania. February 10th

, 2011,

Launceston, Tas, Australia.

Gliniewicz, K, KP Plant, SE LaPatra, KD Cain, KR Snekvik, BR LaFrentz, and DR Call.

Comparative proteomic analysis of virulent and rifampicin attenuated strains of Flavobacterium

psychrophilum. American Fisheries Society Annual Meeting, Seattle, WA, 5-7 September 2011.

Long, A, MP Polinski, DR Call, and KD Cain. Validation of Diagnostic Assays to Screen

Broodstock for Flavobacterium psychrophilum Infection. Talk presented at the Idaho Chapter of

the American Fisheries Society Annual Meeting. Boise, Idaho, March 2-4, 2011.

Swain, MA, A Long, TR Fehringer, BR LaFrentz, DR Call, and KD Cain. Vaccine efficiency in

Coho salmon against Flavobacterium psychrophilum. Talk presented at the Center for Research

on Invasive Species and Small Populations end of summer presentations. Moscow, Idaho,

August 4, 2011.

Long, A, DR Call, and KD Cain. Use of Diagnostic Assays to Screen Rainbow Trout

(Oncorhynchus mykiss) Broodstock for Flavobacterium psychrophilum. Talk presented at the 3rd

Annual Western Division American Fisheries Society Student Colloquium, Moscow, Idaho,

October 14-16, 2010.

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.

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 the WSU

Showcase, March 2010, Pullman, WA.

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Long, A., Call, D.R., and Cain, K.D. 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, 2010.

Gliniewicz, KS, KD Cain, KR Snekvik, and DR 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, Pullman, WA, October 14, 2009.

Long, A, DR Call, and KD Cain. 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, 2009.

SUBMITTED BY: ____________________________________September 8, 2011____ Title: (Work Group Chair or PI) Date

APPROVED: ______ ________September 14, 2011___ Technical Advisor (if Chair’s report) Date

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WRAC TERMINATION REPORT

PART II: Detail

PROJECT TITLE: COLDWATER DISEASE PREVENTION AND

CONTROL THROUGH VACCINE DEVELOPMENT

AND DIAGNOSTIC IMPROVEMENTS

REPORT GIVEN IN YEAR: 2011

PROJECT WORK PERIOD: 5/1/2008-present; no-cost extension approved, 9/30/12)

AUTHOR: Ken Cain and Doug Call

PARTICIPANTS: Kenneth Cain* (Work Group Chair) Univ. of Idaho

Douglas Call* Wash. State Univ.

Scott LaPatra Clear Springs Foods

Gary Fornshell* (Outreach Coordinator) University of Idaho

Greg Weins USDA, West Virginia

Technical Advisor: Gael Kurath USGS, Washington

Industry Advisor: Jim Parsons Troutlodge, Wash.

Graduate Students: Amy Long/Karol Gliniewicz UI/WSU

Faculty participant: Devendra Shah Wash. State Univ.

PROJECT OBJECTIVES:

1. This objective was modified to include studies of both subunit vaccines and the

mechanism responsible for attenuation of strain CSF259.93.B17. This work included

identification and testing of potential subunit vaccine candidates, testing alternative

vaccine delivery strategies, and testing alternative hypotheses regarding the

mechanism(s) responsible for attenuation of the B.17 strain.

2. Validate quantitative diagnostic assays and assess utility for assessing the risk of

vertical transmission. This primary focus included assessing the diagnostic specificity

and sensitivity for diagnostic assays and correlating risk of vertical transmission or

disease susceptibility with pathogen load in brookstock.

3. Develop alternative assays for quantification of infection in ovarian fluid. This work

is focused on development and validation of an alternative real-time PCR assay.

4. Develop an integrated outreach program to meet stakeholder needs. This work

focused on delivery of outreach/extension products related to prevention of coldwater

disease and tailoring management at broodstock facilities.

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TECHNICAL SUMMARY AND ANALYSIS:

Objective 1: The original objective was to identify potential vaccine candidates using

IVIAT, but our strategy later focused on identification of potential subunit vaccines using a

comparative proteomic analysis of the attenuated strain of F. psychrophilum

(CSF259.93.B17) compared to the wild-type strain. With prior approval, some effort was

re-directed to identify the mechanisms responsible for attenuation of CSF259.93.B17.

The attenuated strain of F. psychrophilum (CSF259.93.B17; “B17”) was originally produced by

serial passage on agar plates that contain increasing concentrations of an antibiotic called

rifampicin (LaFrentz et al. 2009). Resistance to this drug is normally conferred by point

mutations in the rpoB gene, which produces a subunit of the RNA polymerase holoenzyme.

Using PCR and sequencing we determined that B17 has a single base-pair mutation in this gene.

Point mutations at this position in the gene (amino acid residue 473 changed from a glutamine to

an arginine) have been correlated with high level resistance to rifampicin and it is highly likely

that this mutation is responsible observed rifampicin resistance for B.17 (Manten and

Wijngaarden 1969, Campbell et al. 2001). Although not studied in detail, we also analyzed a

second partially attenuated strain, CSF259.93.A16, and discovered two base mutations in the

rpoB gene, both of which are located at positions in the DNA sequence that have been correlated

with resistance to rifampicin in other bacteria.

We completed the comparative proteomic analysis for CSF259.93 (wild-type) and attenuated

strains. We used 2-dimensional PAGE to separate proteins from lysed cells after growth in broth

media. This type of electrophoresis allows us to separate proteins on the basis of both molecular

mass and isoelectric point (charge). We identified eight and six proteins that were uniquely up-

regulated in the wild-type and attenuated strains, respectively (Table 1). These include the highly

immunogenic outer membrane antigen P60 (OmpA P60), heat shock protein 70 (DnaK) and

elongation factor Tu (EFTU), which have been examined as possible subunit vaccines (Dumetz

et al. 2007, Plant et al. 2009, 2011). Importantly, these differentially expressed genes are not

grouped in a single locus or operon, which indicates that the attenuation from rifampicin possibly

resulted from a multi-factorial alteration of transcription. We were particularly interested in the

FP1493 because of its apparent responsiveness to iron levels (LaFrentz et al. 2009) and because

it appears to be associated with a HmuY and haemin-uptake gene cluster (hmu) homologous to

Porphyromonas gingivalis. HmuY is a putative haem-binding lipoprotein associated with the

outer membrane, is a part of an operon engaged in haemin utilization and may play a significant

role in biofilm accumulation (Olczak, et al. 2008, Wójtowicz et al. 2009, Olczak et al. 2010). In

addition to iron-acquisition mechanisms suggested by Moller et al. (2005), FP1493 may also be

part of iron-uptake system utilized by F. psychrophilum. If so, FP1493 may be involved in

colonization, iron uptake and growth of F. psychrophilum under iron-limiting conditions within

the host.

Members of outer-membrane family of proteins, OmpA P60 and Omp121 could also be involved

in host-pathogen interactions and play a potential role in protective immunity. OmpA P60, in

particular, was intensively studied in the context of providing protection against F.

psychrophilum (Merle et al. 2003, Dumetz et al. 2007). Additionally, a BLAST search for

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conserved domains reveals similarities of Omp121 with outer-protein families of membrane

channels or TonB-dependent haemoglobin/transferrin/lactoferrin receptors and TonB-dependent

siderophore receptors. Given its putative role in iron acquisition, it may be possible that lack of

Omp121 in the attenuated strain may contribute to its inability to cause disease. The exact

function of peptidyl-proyl cis-trans isomerase (PpiC) in F. psychrophilum has not been

established, but the protein may be a ribosome bound trigger factor or it may be engaged in

protein folding processes (Stoller et al. 1995, Justice et al. 2005, Maier et al. 2005, Kaiser et al.

2006).

Flavo-specific antigen A (FspA) was demonstrated to be immunoreactive with trout convalescent

sera and was tested as a possible subunit vaccine candidate against CWD (Crump et al. 2005).

Involvement of benzil reductase (YueD), FP0216 or Acyl-CoA dehydrogenase protein FP1726 in

modulating host-immune response or virulence is unknown. Interestingly, multiple acyl-CoA

synthases homologs were recently reported to be involved in virulence of Pseudomonas

aeruginosa (Kang et al. 2010). Elongation factor Tu (EF-Tu) that was identified from the wild-

type strain and in E. coli has been reported to play a role in gene expression, DNA repair and

protein processing (Malki et al. 2002). Additionally, EF-Tu was demonstrated to interact with

hydrophobic regions of proteins by assisting refolding in a manner similar to molecular

chaperones such as DnaK/Hsp70 (Caldas et al. 1998, Malki et al. 2002). The wild-type strain

exhibited increased synthesis of the 30S ribosomal subunit S1 encoded by rpsA while the

attenuated strain showed apparent upregulation of the 30S ribosomal subunit S2 (rpsB). These

highly conserved proteins are involved in complex co-regulation and modulation of gene

expression and protein synthesis networks (Wilson and Nierhaus, 2005, Aseev et al. 2008).

Interestingly, in E. coli upregulation of S2 was reported to cause suppression of the tsf gene,

encoding elongation factor-Ts, which is a GDP/GTP exchanger for EF-Tu. With altered function

of EF-Tu, a key player in gene expression, the E. coli mutant overexpressing S2 revealed

defective growth (Aseev et al. 2008). If the same is true for F. psychrophilum, then observed

elevated levels of RpsB in the attenuated strain may be one of possible causes of minor growth

impairment exhibited by this attenuated strain (LaFrentz et al. 2008). Slight growth impairment

was also characteristic for other studies involving rifampicin resistant strains (Moorman and

Mandell 1981, Jin and Gross 1989, Mariam et al. 2004). Another protein with increased spot

intensity found in the rifampicin-attenuated strain was elongation factor G (EF-G, fusA), which is

putatively involved in gene expression and protein synthesis but can also mediate protein folding

and thus exhibit chaperon-like properties (Caldas et al. 2000).

Using a western blot procedure with convalescent antisera, 2-dimensional PAGE, and mass

spectrophotometry, we also identified the antigen that is targeted by our diagnostic antibody,

FL43 (see below). Given the potential role of this protein in iron acquisition and increased

synthesis in vivo, we elected to pursue this protein as a subunit vaccine candidate. We developed

recombinant protein that was expressed using our Vibrio parahaemolyticus heterologous

expression system (Shah et al. 2008). This candidate protein has additional desirable properties

including ease of expression and solubility making purification relatively simple. Furthermore,

while the protein has a relatively low mass (≈23 kDa), it forms multimers of sufficient size to be

found in the upper size fraction that LaFrentz et al. (2004) identified as being important for

humoral protection against challenge by F. psychrophilum. Two immunization and challenge

trials were conducted and the recombinant FP1493 was administered with adjuvant (Freund’s

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complete adjuvant) following our standard protocols (LaFrentz et al. 2004). While this procedure

produced significantly elevated titers against FP1493, we found no evidence of protection

against an injection challenge with CSF259.93 (data not shown). A paper describing this work

has been submitted for peer-review.

Our working hypothesis is that CSF259.93.B17 is attenuated because the mutation in the rpoB

gene interferes with the ability of the RNA polymerase holoenzyme to bind to promoter

sequences or it interferes with the polymerase interaction with sigma factors. To test this

hypothesis we are in the process of generating a “knock-in” mutant. This involves replacing the

wild-type rpoB sequence with one that includes the same point mutation that we have

documented for the attenuated strain. As a general rule, genetic manipulation of F.

psychrophilum is difficult at best and to date has been impossible with our virulent strain,

CSF259.93. Consequently, we are in the process of producing the “knock-in” strain using an

alternative strain that we have shown is highly virulent by injection challenge (unpub. data), but

that is also amenable to genetic manipulation. If the proteomic profile and attenuation of

virulence are recapitulated by the knock-in experiment, this will support the hypothesis that

attenuation results from the mutated rpoB. We have acquired or developed the necessary

reagents for this experiment in conjunction with our collaborator, Dr. Mark McBride (Univ.

Wisconsin). The appropriate homologous exchange vector has been constructed and we are in

the process of generating the knock-in strain (data not shown).

In the past year we were also able to conduct a 454 sequencing experiment of the wild-type and

attenuated strains. This experiment (which was completed at no additional charge to the project)

identified 24 nonsynonymous mutations in the genome of the attenuated strain (there were only 8

for the wild-type strain). This high degree of mutation raises the possibility that attenuation

results from mutations in genes other than rpoB, although we cannot ascertain if the mutation

process was due to long-term selection on agar plates or due to the influence of rifampicin

selection pressure. The uncertainty about the cause of mutation arises because we did not have a

wild-type strain that was co-passaged with the B.17 strain, but without antibiotic selection

pressure. We have now completed new passage experiments generating two rifampicin-resistant

strains in two different backgrounds. DNA has been extracted and we are awaiting sequence data

from Illumina before we can proceed with identifying single-nucleotide polymorphisms.

Resources permitting we will also determine if the new rifampicin-resistant strains are also

attenuated for injection challenge in rainbow trout.

CSF259.93.B.17 Efficacy in Coho Salmon. The live attenuated vaccine for F .psychrophilum

(B.17) was originally tested in rainbow trout (LaFrentz et al. 2008). As BCWD can affect several

species of salmonids, we recently tested the efficacy of the vaccine in Coho salmon. We also

sought to improve the effectiveness of the vaccine by altering the growth media. Previous work

in our lab has shown that F. psychrophilum has increased protein expression when grown in iron-

limited media as well as increased antigen expression on the exterior of the cell (LaFrentz, et al.

2009). We hypothesized that growing the 259-93 B.17 strain in iron-limited media would result

in increased immunogenicity leading to increased protection in fish when challenged with the

virulent wild-type strain. F. psychrophilum strains were cultured in either TYES or TYES with

50 µM of 2,2-bipyridyl (DPD), an iron chelator, and harvested for vaccinations and disease

challenge using previously published protocols (LaFrentz et al. 2002; LaFrentz et al. 2009).

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Two different delivery methods, immersion and injection, were also tested. Fish were either

vaccinated with an intraperitoneal injection, or adipose fin clipped and immersed for 1 hr in the

vaccine at a concentration of 1 part vaccine to 3 parts tank water. Fish were booster immunized

at 4 weeks post-immunization using the same delivery methods. Antibody titers in immunized

fish were measured using the ELISA protocol developed by LaFrentz et al (2002). Serum was

collected prior to immunization and again at 4, 6, and 12 weeks post-immunization. At 6 weeks

post-immunization, triplicate groups of 25 fish from each treatment were subcutaneously injected

with the F. psychrophilum CSF259-93. In each group, a subset of fish (n=20) were injected with

sterile PBS to serve as the mock infected control. Mortalities were monitored on a daily basis for

28 days and re-isolation of F. psychrophilum was attempted by sampling 20% of the daily

mortalities and streaking kidney, liver, and spleen samples on TYES agar and incubating plates

at 15°C for 96 hr. The cumulative percent mortality (CPM) and relative percent survival (RPS)

were calculated for each treatment group. RPS was calculated using the following formula:

(1 – (CPM vaccinated group/CPM unvaccinated group)) x 100

Differences in CPM and antibody titer (log10 transformed) were determined using a 1-way

ANOVA. If the differences were significant, then Tukey's post-hoc test was carried out to

determine which groups were different.

In the injection trial, antibody titers were significantly higher in both treatment groups than the

control at all time points (P<0.0001) (Table 2). The difference in titers between the two

treatment groups was not significant. A similar trend was noted for fish in the immersion group.

F. psychrophilum was re-isolated from 91% of the mortalities examined. In the injection trial, the

CPM was significantly lower in the immunized groups as compared to the mock immunized

group (P<0.05) (Table 1). In the immersion trial, only the CPM of the group immersed in 259-93

B.17 w/ DPD was significantly lower than the control (P<0.05). Overall, RPS was higher in the

injection group than in the immersion. However, for both delivery methods, the RPS was highest

in fish immunized with 259-93 B.17 w/ DPD. Based on these results, we can conclude that the

live attenuated CSF 259-93 B.17 strain previously shown to protect rainbow trout against BCWD

is also effective in Coho salmon. While it is not statistically significant, it appears that the B.17

strain grown in iron limited media may confer increased protection compared to the B.17 strain

grown in regular TYES.

Objective 2: Validate quantitative diagnostic assays and assess utility for assessing the risk

of vertical transmission.

Assay Validation. Protocols for both the ELISA and MF-FAT were finalized in 2009 and are

now available upon request as well as on the ImmunoPrecise Antibodies, Ltd. website.

Validation of the assays has been an ongoing process that was recently completed. To determine

if the ELISA could be used to screen individual broodstock, adult rainbow trout were injected

with 2.7 x 107 CFU fish

-1 of F. psychrophilum CSF 259-93. All kidney samples collected at 7

days post-injection and one kidney sample taken at 10 days post-injection had ELISA O.D.

values above the detection threshold. The results of this experiment indicate the ELISA could

indeed be used for screening individual fish that had been exposed to F. psychrophilum. The next

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step in validating the assays was estimation of diagnostic sensitivity (the proportion of positive

samples that are correctly identified as positive), analytical sensitivity (lowest level of antigen

that can be detected), and specificity (proportion of negative samples that are correctly identified

as negative). The kidney tissue ELISA has a sensitivity of 0.97 and specificity of 0.98. In

contrast, the ovarian fluid MF-FAT sensitivity is 0.25 and specificity is 0.02. The glycocalyx

layer of F. psychrophilum is easily disrupted during the filtration process which would limit the

ability of MAb FL43 to bind to the cells during the MF-FAT procedure (Vatsos et al. 2002).

Finally, FP1493, the outer membrane protein reactive to MAb FL43, has increased expression

when grown in vivo and in iron-limited media which is suspected to mimic the natural host

environment (LaFrentz et al. 2009). As such, we hypothesized that analytical sensitivity of the

ELISA would increase when using F. psychrophilum cells grown in iron-limited media. To test

this, DPD was added to TYES media at a final concentration of 50, 62.5, and 75 µM. Addition of

DPD results in decreased growth but increased outer membrane protein expression leading to an

increase in analytical sensitivity. The ELISA is able to detect as few as 280 F. psychrophilum

cells from a broth culture grown in TYES with 75 µM DPD.

Broodstock infection levels and progeny outbreaks. To establish a biologically relevant assay

for control of BCWD control, it is essential to correlate risk of vertical transmission or disease

susceptibility in progeny with assay results from broodstock. To accomplish this, two separate

trials were carried out in 2010 and 2011. In the first, 60 female rainbow trout broodstock from

Troutlodge were sampled in February 2010. In the second trial, 30 Coho salmon returning to

Skookum Creek Hatchery were sampled in November 2010. In both trials, kidney, spleen, and

ovarian fluid were collected and used in the ELISA, MF-FAT, nested PCR and bacteriological

culture. Fertilized eggs from the sampled fish were incubated separately until the eyed stage at

which point progeny from five broodstock deemed to have varying levels of infection were sent

to University of Idaho. Results for the broodstock used in the two trials are listed in Table 3.

None of the kidney samples from Troutlodge were positive by ELISA and only three spleen

samples were above the detection threshold while 100% of Skookum Creek broodstock had

positive kidney samples and 17% had positive spleen samples.

In both the rainbow trout and Coho salmon trials, progeny were kept separate by family

throughout all experiments. In the first set of experiments, progeny were sampled on a regular

basis from arrival at UI as eyed eggs until approximately 2 months after arrival to determine if

there were any family differences in detection of F. psychrophilum during this time period. For

the rainbow trout trial, samples were collected weekly from each family, pooled, and DNA then

extracted from the pooled sample using Qiagen DNeasy kits. Nested PCR was then done using

the DNA to check for presence/absence of the bacterium (Taylor 2004). F. psychrophilum was

detected in material sampled from eyed eggs that had been disinfected in 400 ppm Ovadine® for

15 min. Detection of the bacteria in samples appeared to decrease as the fish grew and by 80

days post-arrival, the bacterium was no longer detected in pooled samples (Table 4).

For the Coho trial, eggs were sampled individually upon arrival at UI. Individual sampling

continued throughout this period and samples were taken twice a week for 50 days. The

proportion of total positive samples was then determined for each family on each sampling date.

Once again, F. psychrophilum was detected in disinfected eyed eggs upon arrival at UI. In

contrast to the rainbow trout trial, the proportion of samples testing positive increased throughout

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the monitoring period (Fig. 1). While the proportion of positive samples in each family was not

significantly different, we did observe that F20 and F30, the families with the highest kidney

ELISA O.D. values, also had the highest total proportion of positive samples, 0.48 and 0.45,

respectively.

In summary, the results of the progeny monitoring experiments indicate that vertical

transmission of F. psychrophilum occurred in both trials. Furthermore, the increase in proportion

of positive samples in the Coho salmon population indicates there was horizontal transmission of

the bacterium resulting in high frequency of detection and increased risk for outbreaks.

When progeny reached the desired weight (0.5 g in rainbow trout and 0.3 g in Coho salmon),

stress experiments were initiated. Stress experiments were done in an attempt to induce a natural

BCWD outbreak in the population and correlate frequency of outbreaks to infection level in the

broodstock. In the rainbow trout trial, 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. As

F. psychrophilum can be present without any clinical signs of disease, 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 (TYES plus 5 µg ml-1

tobramycin) and sub-samples taken for nested PCR.

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 (Figure 2). While we did not

have a full-scale outbreak of BCWD, we did show an increase in F. psychrophilum in the

population being studied. Fish exhibited signs of chronic stress including frayed fins, petechial

hemorrhaging, scale loss, and overinflated swim bladders. Additionally, 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 nested PCR showed that F. psychrophilum was present in 29.6% of mortalities.

F. psychrophilum was not re-isolated by bacteriological culture.

In the Coho salmon stress challenge in trial 2, stress experiments were started one week after

initiation of feeding when fish weighed approximately 0.3 g. The stressors chosen for this

challenge were picked to mimic conditions in Skookum Creek hatchery where outbreaks of

BCWD are common. There were three control and three treatment tanks per family. Seventy-five

fry were stocked into each tank. To achieve different stocking densities, the volume of the tanks

was modified and was either 17.5 L (control) or 4 L (treatment, high stocking density). The flow

rate in the treatment tanks was 0.2 L min-1

while the flow rate in the control tanks was 2.5 L min-

1. Fish were maintained at these conditions for 8 weeks and monitored for disease symptoms.

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Samples were taken from each tank twice a week and checked for F. psychrophilum. In total,

there were 21 mortalities during this experiment with no difference in mortalities between

families. However, several mortalities (n=4) from both treatment and control tanks did exhibit

the classic symptoms of F. psychrophilum including dorsal surface erosion and missing caudal

fin. Weekly samples and bacterial isolates from this challenge are still being processed.

However, YPB bacteria with the fried egg morphology were commonly re-isolated from

homogenized fish during this experiment indicating that F. psychrophilum was present in the

population. In both trials, we were unable to induce a BCWD outbreak in the experimental

population when reared under stressful conditions. We hypothesize that this failure to induce an

outbreak is linked to numerous abiotic and biotic factors found in the hatchery setting that may

influence the risk of outbreaks in fry but cannot be replicated in the laboratory environment.

The final and third experiment done with progeny collected from broodstock with varying

infection levels looked at progeny susceptibility to F. psychrophilum when directly challenged

with the bacterium. Fish (mean weight 5 g) were subcutaneously injected with a virulent strain of

F. psychrophilum strain CSF 259-93 using our standard challenge procedures (LaFrentz et al.

2002). Due to mechanical issues in the wetlab facility, the challenge was not completed in the

rainbow trout trial and no data was collected. The challenge is currently ongoing with Coho

salmon. Due to space constraints, the first two families, F6 and F19, were challenged separately

from the remaining families. In this challenge, no mortalities were noted in the low dose (6.6 x

105 CFU fish

-1) for either family. As a result, the concentration of the low dose was increased to

9.4 x 105 CFU fish

-1 in the second challenge. Mortalities in the low dose for F20, F27, and F30

have been noted but the difference in survival for these three families is not significant. The high

dose was kept constant between the two challenges (106 CFU fish

-1). Preliminary results indicate

that there is a significant difference between families as determined by log-rank analysis of the

survival curves from the high dose (Figure 3). Specifically, F27 had significantly lower survival

than the other families. F27 broodstock had one of the lowest kidney ELISA O.D. values but one

of the higher spleen O.D. values. It is possible that genetic differences between families are

influencing progeny susceptibility. Previous research has shown that there is a family related

susceptibility to F. psychrophilum in direct challenges (Johnson, Vallejo, Silverstein, Welch,

Wiens, Hallerman & Palti 2008; Silverstein, Vallejo, Palti, Leeds, Rexroad, Welch, Wiens &

Ducrocq 2009). However, we will collect serum from challenge survivors to determine if there

are differences in specific F. psychrophilum antibody titer between families.

As part of this project, we have also collected samples from 6 hatcheries (state, tribal, federal,

and private). The purpose of this is two-fold. In addition to validating the ELISA and other

diagnostic assays, we have also been able to look at differences in prevalence between

anadromous fish and hatchery reared fish. While that data is still being compiled, early results

indicate that prevalence in broodstock is overall higher at hatcheries spawning anadromous fish.

At Wallowa State Hatchery, prevalence of F. psychrophilum in steelhead kidney samples was

0.23 while the prevalence in the ovarian fluid samples was estimated to be 0.33 for samples

collected in Spring 2010. At Dworshak National Fish Hatchery, the estimated prevalence for

steelhead samples from Spring 2009 was the same for both sample types, 0.97. Conversely, at

Troutlodge, the estimated prevalence in both kidney tissue and ovarian fluid was low, 0.016 and

0.12 respectively.

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In conclusion, we have not been able to successfully link broodstock infection levels to risk of

progeny outbreaks in either population. Nevertheless, we have shown that the ELISA can be

used to determine antigen levels in populations which would allow for screening of broodstock

and development of a herd health management program. Progeny from broodstock with higher

antigen levels or those that are positive by multiple assays may be more likely to carry F.

psychrophilum resulting in increased horizontal transmission of the bacterium during stressful

periods and an increased risk of outbreaks.

Objective 3: Develop alternative assays for quantification of infection in ovarian fluid.

Development of a quantitative PCR assay for F. psychrophilum detection in ovarian fluid and

tissue samples has continued. Due to the nature of ovarian fluid, one of the biggest hurdles in

development of the assay has been optimization of DNA extraction and reducing loss of target

during the extraction process. These are currently being optimized and it is thought that once we

have finalized this protocol which should be shortly, the assay will move into testing archived

samples and comparing to other assay results.

In addition, we have worked with Infoscitex, Inc. on developing new diagnostic assays for tissue

and ovarian fluid. Infoscitex was awarded a SBIR grant from USDA to develop an aptamer assay

for F. psychrophilum. Aptamers are single-stranded DNA or RNA oligonucleotides that are able

to bind viruses, proteins, and small molecules. Phase I of this grant has been completed.

Infoscitex developed four possible aptamers against the outer membrane of F. psychrophilum.

Targets were sent to UI in April 2011 where quantitative PCR assays with these targets were

successfully optimized. This information has been communicated to Infoscitex and a request for

Phase II funding will most likely be submitted in 2012.

Objective 4: Develop an integrated outreach program to meet stakeholder needs.

Outreach activities have resulted in two articles in Waterlines describing this WRAC project and

additional information highlight in the 2011 fall issue of Trout Talk. There have been media

releases and reports on our vaccine work along with numerous presentations at professional and

other meetings. In summer 2011, we participated in the University of Idaho's Center for

Research on Invasive Species and Small Populations (CRISSP) Research Experience for

Undergraduates (REU) program (and NSF funded program). An undergraduate from Eckerd

College was selected to work on the CSF 259-93 B.17 vaccine efficacy in Coho salmon project.

In addition to gaining valuable lab experience, the undergraduate student also presented the

results of her project to her peers at the conclusion of the program. Additionally, in July 2011,

we led a workshop at the Salmon Disease Course at Oregon State University. During the day-

long workshop, participants carried out an ELISA with MAb FL43 using kidney from fish

injected with F. psychrophilum. Participants in the course represented state, provincial, tribal,

and federal agencies as well as industry including Schering-Plough and Marine Harvest.

Additional WRAC publications will be developed following completion of current studies.

Outreach activities will be a primary focus over the next year and beyond. Currently, the

patented vaccine is under field evaluation through a partnership with Aquatic Life Sciences who

has signed an option agreement with UI to licensing the patent. If results are promising, it is

expected that the vaccine could be commercialized and sold under a USDA conditional license

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approval as early as January 2012. If commercialization of this vaccine occurs it may then be

possible to determine long term impact due to adoption and implementation of a vaccine to

control CWD and subsequent reduction of mortalities due to CWD. A survey of the target

audience or aquaculture vaccine manufacturers may provide the information needed for long

term impact evaluation. The results of the initial vaccine field trial will be presented to the

Pacific Northwest Fish Health Protection Committee annual meeting and the joint US Trout

Farmers Association and Idaho Aquaculture Association Fall Conference in September 2011. If

the vaccine is effective, a downloadable WRAC outreach publication that will briefly cover

(there’s already a WRAC CWD publication) CWD, what it is, how to diagnosis it and its impact

and then in great detail how to use the vaccine and expected results based on the

immunization/challenge and field results. In addition, a section will be added to the WRAC

outreach publication describing the methodology of the diagnostic tools, how to apply the tools

for rapid CWD diagnosis and/or implementation of a broodstock and/or egg culling program.

The results would provide effective early detection of disease and treatment of juvenile fish and

possible long term reduction of disease if culling programs are implemented. The group will

follow-up with ImmunoPrecise and fish health labs to quantify impacts through the sale and use

of the monoclonal antibody FL43. One to two years after project completion a survey of the

target audience will attempt to determine the extent of diagnostic tool use and if broodstock

and/or egg culling programs are used to minimize CWD outbreaks. If the vaccine is

commercialized then workshops for private and public salmonid hatchery personnel will be held

to explain and demonstrate how to use the vaccine and incorporate the diagnostic tool for early

detection of the disease. An impact statement will be written after an evaluation of the

deliverables to industry and other stakeholders

FOLLOW UP ACTIVITIES: We will complete the experiments outlined above to determine

the mechanism that is responsible for attenuation of CSF259.93.B17. As previously noted,

hatcheries spawning anadromous salmonids appear to have a higher prevalence of F.

psychrophilum than rainbow trout rearing facilities. As such, we intend to sample rainbow trout

broodstock at Troutlodge at least one more time to evaluate prevalence in these broodstock. We

would also like to sample at least one more anadromous fish hatchery. This data could be useful

in the long run in reducing risks of outbreaks at hatcheries especially if vertical transmission is

more of a factor at hatcheries spawning returning broodstock than rainbow trout facilities (Chen

et al. 2008).

Based on the results of the analytical sensitivity experiments and the vaccine trial with Coho

salmon, work is scheduled to begin shortly evaluating a waterborne challenge model for F.

psychrophilum using bacterium that has been grown in iron-limited media. We hypothesize that

bacteria grown in iron-limited media will have increased pathogenicity due to the increased and

novel protein expression. Pathogen-free rainbow trout will be immersed in F. psychrophilum

grown in iron-limited media as well as regular media and mortalities monitored. A sub-sample of

fish will be adipose fin clipped to determine if this enhances successful infection. An immersion

challenge model has long been desired as it is thought be representative of the natural route of F.

psychrophilum. If we are able to successfully develop one, this will allow for better evaluation of

disease prevention strategies such as vaccination and immune stimulants.

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IMPACTS:

Relevance: There is a strong need for public and private aquaculture facilities to have additional

control and management options for CWD.

Response: One deliverable from this project 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. Diagnostic

assays to cull infected broodstock are established and 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 and they

are available as downloadable pdfs directly from their website. The other deliverable will

hopefully be a commercialized vaccine for CWD. The B.17 vaccine was patented by the

University of Idaho in June 2010 and is currently being field tested for efficacy. A UI press

release about the vaccine was sent to a broad array of stakeholders including Idaho trout growers.

Recent experiments showing enhanced protection of the B.17 vaccine are viewed as potential

enabling technology and a provisional patent application was filed in August, 2011 to protect

improved methodology.

Results: Impacts of the deliverables will be evaluated in follow-up activities (see below).

Impacts: Currently, the patented vaccine is under field evaluation through a partnership with

Aquatic Life Sciences who has signed an option agreement with UI to licensing the patent. If

results are promising, it is expected that the vaccine could be commercialized and sold under a

USDA conditional license approval as early as January 2012. If commercialization of this

vaccine occurs it may then be possible to determine long term impact due to adoption and

implementation of a vaccine to control CWD and subsequent reduction of mortalities due to

CWD. A survey of the target audience or aquaculture vaccine manufacturers may provide the

information needed for long term impact evaluation. The results of the initial vaccine field trial

will be presented to the Pacific Northwest Fish Health Protection Committee annual meeting and

the joint US Trout Farmers Association and Idaho Aquaculture Association Fall Conference in

September 2011. If the vaccine is effective, a downloadable WRAC outreach publication that

will briefly cover (there’s already a WRAC CWD publication) CWD, what it is, how to

diagnosis it and its impact and then in great detail how to use the vaccine and expected results

based on the immunization/challenge and field results. In addition, a section will be added to the

WRAC outreach publication describing the methodology of the diagnostic tools, how to apply

the tools for rapid CWD diagnosis and/or implementation of a broodstock and/or egg culling

program. The results would provide effective early detection of disease and treatment of

juvenile fish and possible long term reduction of disease if culling programs are implemented.

The group will follow-up with ImmunoPrecise and fish health labs to quantify impacts through

the sale and use of the monoclonal antibody FL43. One to two years after project completion a

survey of the target audience will attempt to determine the extent of diagnostic tool use and if

broodstock and/or egg culling programs are used to minimize CWD outbreaks. If the vaccine is

commercialized then workshops for private and public salmonid hatchery personnel will be held

to explain and demonstrate how to use the vaccine and incorporate the diagnostic tool for early

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detection of the disease. An impact statement will be written after an evaluation of the

deliverables to industry and other stakeholders.

Collaborators: Progress in the area of vaccine development and diagnostic improvements

supported in part through this WRAC project would not have been possible without efforts of

many collaborators. Company involvement by Troutlodge and Clear Spring’s Foods, Inc. were

vital to evaluation and sample collection from private industry. Additionally, we are indebted to

all the assistance provided by the various agency personnel (Northwest Indian Fisheries

Commission; Washington Department of Wildlife and Fisheries; Oregon Department of Fish and

Wildlife; etc.) who provided samples of Coho and Steelhead for testing.

PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED:

Refereed publications:

Plant, KP, SE LaPatra, DR Call, and Cain, KD. Immunization of rainbow trout (Oncorhynchus

mykiss) with Flavobacterium psychrophilum gliding motility protein N. Journal of Fish

Diseases (In review)

Long, A, MP Polinski, DR Call, and KD Cain. Validation of diagnostic assays to screen

broodstock for Flavobacterium psychrophilum infections. Journal of Fish Diseases (In review)

Gliniewicz, K, KP Plant, SE LaPatra, BR LaFrentz, K Cain, KR Snekvik and DR Call.

Comparative proteomic analysis of virulent and rifampicin attenuated Flavobacterium

psychrophilum. Journal of Fish Diseases (In review)

LaFrentz, B.R., LaPatra, S.E., Call, D.R., Wiens, G.D., and Cain, K.D. 2011. Identification of

Immunogenic proteins within distinct molecular mass fractions of Flavobacterium

psychrophilum. Journal of Fish Diseases (In Press)

Plant, KP, SE LaPatra, DR Call, and KD Cain. 2011. Immunization of rainbow trout

(Oncorhynchus mykiss) with Flavobacterium psychrophilum proteins elongation factor-Tu, SufB

Fe-S assembly protein and ATP synthaseβ. Journal of Fish Diseases 34, 247-250

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

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General articles:

Cain, KD. 2009. Strategies for Control and Prevention of Coldwater Disease. Waterlines

newsletter 15 (1): p. 18-20.

Cain, KD and DR Call. 2010. Coldwater disease. Waterlines Newsletter, Spring 2010, p10.

Cain, K and DR Call. Coldwater Disease Research. Trout Talk, Fall, 2011.

Presentations:

Cain et al. A potential vaccine to control bacterial coldwater disease. US Trout Farmers

Association and Idaho Aquaculture Association Fall Conference. Twin Falls, ID. Sept. 29-Oct.

1, 2011.

Cain and Zinn. BCWD Vaccine Development. 56th Pacific Northwest Fish Health Protection

Committee Annual Meeting, Portland, OR. Sept. 21-22, 2011.

Cain, K.D. Research overview and update. University of Tasmania. February 10th

, 2011,

Launceston, Tas, Australia.

Gliniewicz, K, KP Plant, SE LaPatra, KD Cain, KR Snekvik, BR LaFrentz, and DR Call.

Comparative proteomic analysis of virulent and rifampicin attenuated strains of Flavobacterium

psychrophilum. American Fisheries Society Annual Meeting, Seattle, WA, 5-7 September 2011.

Long, A, MP Polinski, DR Call, and KD Cain. Validation of Diagnostic Assays to Screen

Broodstock for Flavobacterium psychrophilum Infection. Talk presented at the Idaho Chapter of

the American Fisheries Society Annual Meeting. Boise, Idaho, March 2-4, 2011.

Swain, MA, A Long, TR Fehringer, BR LaFrentz, DR Call, and KD Cain. Vaccine efficiency in

Coho salmon against Flavobacterium psychrophilum. Talk presented at the Center for Research

on Invasive Species and Small Populations end of summer presentations. Moscow, Idaho,

August 4, 2011.

Long, A, DR Call, and KD Cain. Use of Diagnostic Assays to Screen Rainbow Trout

(Oncorhynchus mykiss) Broodstock for Flavobacterium psychrophilum. Talk presented at the 3rd

Annual Western Division American Fisheries Society Student Colloquium, Moscow, Idaho,

October 14-16, 2010.

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.

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 the WSU

Showcase, March 2010, Pullman, WA.

Long, A., Call, D.R., and Cain, K.D. 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, 2010.

Gliniewicz, KS, KD Cain, KR Snekvik, and DR 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, Pullman, WA, October 14, 2009.

Long, A, DR Call, and KD Cain. 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, 2009.

SUBMITTED BY:______________________________________September 8, 2011____ Title: (Work Group Chair or PI) Date

APPROVED: _ ____________September 14, 2011____ Technical Advisor (if Chair’s report) Date

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Table 1. Proteins differentially expressed in the parent and attenuated F. psychrophilum strains

as identified by LC-MS/MS. Spot numbers refer to proteins in original 2-dimensional gels (not

shown); pI – isoelectric point.

Spot

numbera

Putative protein

identity

NCBI

access

numberb

Predicted

mass (kDa) /

pI

Peptides matched

(sequence

coverage)

Mascot

scorec

1WT EFTU FP1184 43.2 / 5.14 6 (29%) 1803

2WT FspA FP2019 21.3 / 9.35 4 (38%) 188

3WT Omp 121 family outer

membrane protein

FP1199 115 / 8.94 7 (15%) 91

4WT Protein of unknown

function

FP1493 22.7 / 8.61 5 (50%) 1860

5WT RpsA FP1793 75.7 / 5.72 6 (17%) 183

6WT Protein of unknown

function

FP1496 36.3 / 8.77 7 (35%) 156

7WT PpiC FP1908 33.5 / 6.02 6 (38%) 100

8WT YueD FP1165 27.8 / 7.68 4 (16%) 64

9B17 OmpA (P60) FP0156 49.9 / 4.87 5 (16%) 108

10B17 DnaK / Hsp70 FP0864 67.3 / 4.83 13 (32%) 626

11B17 RpsB FP0454 30.9 / 8.92 8 (35%) 392

12B17 FusA FP1341 79.4 / 5.12 15 (56%) 122

13B17 Protein of unknown

function

FP0261 34.8 / 8.46 4 (21%) 526

14B17 Acyl-CoA

dehydrogenase family

protein

FP1726 66.2 / 5.17 6 (13%) 50

a Subscripts of spot numbers indicate: WT = CSF 259.93 and B17 = CSF253.93B.17 strains.

b Accession number for F. psychrophilum genome sequence.

c A higher Mascot score represents greater confidence in the predicted protein match.

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Table 2. Coho salmon vaccine trial results. Antibody titers and mortality data for the two different delivery methods are listed. *

denote titers from injection immunized fish that are significantly greater than the control group. # denote titers from immersion

immunized fish that are significantly greater than the control group. ^ denotes CPM in treatment groups that are significantly less than

the control. § denotes CPM in immersion immunized fish that are significantly less than the control group.

Delivery method Treatment Ab Titer

4 weeks

Ab Titer

6 weeks

Ab Titer

12 weeks CPM RPS

Injection

PBS 40 ± 7 40 ± 7 200 ± 55 65

259-93 B.17 800 ± 278* 2720 ± 697* 8960 ± 1568* 7^ 90

259-93 B.17 w/

DPD 490 ± 90* 1640 ± 374*

14720 ±

4703* 1

^ 98

Immersion

TYES < 50 < 50 140 ± 24 54

259-93 B.17 1480 ± 315# 1760 ± 261

# 4480 ± 784* 29 47

259-93 B.17 w/

DPD 1680 ± 278

# 880 ± 80

# 5440 ± 1998* 15

§ 73

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Table 3. Assay results for broodstock selected for progeny experiments.

Tissue Samples Ovarian Fluid Culture

Trial Family Kidney

ELISA

Spleen

ELISA nPCR

MF-

FAT Confirmed

Rainbow

Trout

F54 - - - +

F61 - 0.115 - + Kidney

F70 - 0.124 - +

F74 - 0.117 - +

F87 - - - + Spleen, OF

Coho

Salmon

F6 0.094 - - + -

F19 0.100 - - + .

F20 0.133 0.131 - + OF

F27 0.096 0.143 - + OF

F30 0.127 0.140 - + .

Table 4. Summary of results from weekly monitoring of progeny in trial 1. (-) = negative; (+) =

positive; NS= no sample.

Sampling Days

Family Assaya

0b

9 16 23 29 36 44 51 57 64 72

F54 nPCR + - + + + - - + NS NS NS

YPB - - - - - - + + - - -

F61 nPCR + + + + - - - - NS NS NS

YPB - - - - - - - - - - -

F70 nPCR + - + + + - - - NS NS NS

YPB - - - - - + + - - - -

F74 nPCR + + - + + - - - NS NS NS

YPB - - - - - + + + - - -

F87 nPCR + + - + - - - - NS NS NS

YPB - - - - - + - - - - - a nPCR = nested PCR; YPB = yellow pigmented bacteria

b This time point represents samples taken from eyed eggs following disinfection.

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Figure 1. Total proportion of F. psychrophilum positive samples in each family during the Coho

salmon progeny experiment. Multiple samples were collected from each family and the

proportion calculated. The increase with time indicates horizontal transmission of the bacterium

was occurring.

Figure 2. Proportion of F. psychrophilum positive samples from the rainbow trout stress

experiment.

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Figure 3. Survival curves for progeny from Coho salmon broodstock with varying infection

levels directly challenged with F. psychrophilum. * denotes that F27 has significantly lower

survival than the other four families in the high dose treatment.

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