university of washington - wrac termination report project … · 2019. 6. 21. · project...
TRANSCRIPT
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
2
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
3
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.
4
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
5
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
6
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.
7
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.
8
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
1
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.
2
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
3
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
4
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).
5
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
6
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
7
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.
8
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.
9
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
10
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.
11
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
12
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
13
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
14
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
15
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.
16
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
17
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.
18
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.
19
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.
20
References Cited:
Aseev L.V., Levandovskaya A.A., Tchufistova L.S., Scaptsova N.V. and Boni I.V. (2008) A new
regulatory circuit in ribosomal protein operons: S2-mediated control of the rpsB-tsf
expression in vivo. RNA, 14, 1882-1894.
Caldas T.D., El Yaagoubi A. and Richarme G. (1998) Chaperone properties of bacterial
elongation factor EF-Tu. Journal of Biological Chemistry, 273, 11478-82.
Caldas T., Laalami S. and Richarme G. (2000) Chaperone properties of bacterial elongation
factor EF-G and initiation factor IF2. Journal of Biological Chemistry, 275, 855-860.
Campbell E.A., Korzheva N., Mustaev A., Murakami K., Nair S., Goldfarb A., Darst S.A. (2001)
Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell, 104,
901-912.
Chen, Y.C., Davis, M.A., LaPatra, S.E., Cain, K.D., Snekvik, K.R. & Call, D.R. (2008) Genetic
diversity of Flavobacterium psychrophilum recovered from commercially raised rainbow
trout, Oncorhynchus mykiss (Walbaum), and spawning Coho salmon, O. kisutch
(Walbaum). Journal of Fish Diseases, 31, 765-773.
Crump E.M., Burian J., Allen P.D. and Kay W.W. (2005) Identification and expression of a host-
recognized antigen, FspA, from Flavobacterium psychrophilum. Microbiology, 151,
3127–3135.
Dror, M., Sinyakov, M.S., Okun, E., Dym, M., Sredni, B. & Avtalion, R.R. (2006) Experimental
handling stress as infection-facilitating factor for the goldfish ulcerative disease.
Veterinary Immunology and Immunopathology, 109, 279-287.
Dumetz F., LaPatra S.E., Duchaud E., Claverol S. and Le Henaff M. (2007) The Flavobacterium
psychrophilum OmpA, an outer membrane glycoprotein, induces a humoral response in
rainbow trout. Journal of Applied Microbiology 103, 1461–1470.
Jin D.J. and Gross C.A. (1989) Characterization of the pleiotropic phenotypes of rifampin-
resistant rpoB mutants of Escherichia coli. Journal of Bacteriology, 171, 5229-5231.
Johnson, N.A., Vallejo, R.L., Silverstein, J.T., Welch, T.J., Wiens, G.D., Hallerman, E.M. &
Palti, Y. (2008) Suggestive association of major histocompatibility IB genetic markers
with resistance to bacterial cold water disease in rainbow trout (Oncorhynchus mykiss).
Marine Biotechnology, 10, 429-437.
Justice S.S., Hunstad D.A., Reiss Harper J., Duguay A.R., Pinker J.S., Bann J., Frieden C.,
Silhavy T.J. and Hultgren S.J. (2005) Periplasmic peptidyl prolyl cis-trans isomerases are
not essential for viability, but SurA is required for pilus biogenesis in Escherichia coli.
Journal of Bacteriology, 187, 7680–7686.
Kaiser C.M., Chang H-C, Agashe V.R., Lakshmipathy S.K., Etchells S.A., Hayer-Hartl M., Hartl
F.U. and Barral J.M. (2005) Real-time observation of trigger factor function on
translating ribosomes. Nature, 444, 455-460.
Kang Y., Zarzycki-Siek J., Walton C.B., Norris M.H. and Hoang T.T. (2010) Multiple FadD
acyl-CoA synthetases contribute to differential fatty acid degradation and virulence in
Pseudomonas aeruginosa. PLoS One, 5, e13557 1-17.
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.
21
LaFrentz, B.R., LaPatra, S.E., Call, D.R. & Cain, K.D. (2008) Isolation of rifampicin resistant
Flavobacterium psychrophilum strains and their potential as live attenuated vaccine
candidates. Vaccine, 26, 5582-5589.
LaFrentz, B.R., LaPatra, S.E., Call, D.R., Wiens, G.D. & Cain, K.D. (2009) Proteomic analysis
of Flavobacterium psychrophilum cultured in vivo and in iron-limited media. Diseases of
Aquatic Organisms, 87, 171-182.
LaFrentz, B.R., LaPatra, S.E., Jones, G.R., Congleton, J.L., Sun, B. & Cain, K.D. (2002)
Characterization of serum and mucosal antibody responses and relative per cent survival
in rainbow trout, Oncorhynchus mykiss (Walbaum), following immunization and
challenge with Flavobacterium psychrophilum. Journal of Fish Diseases, 25, 703-713.
Maier T., Ferbitz L., Deuerling E. and Ban N. (2005) A cradle for new proteins: trigger factor at
the ribosome. Current Opinion in Structural Biology 15: 204-212.
Malki A., Caldas T., Parmeggiani A., Kohiyama M. and Richarme G. (2002) Specificity of
elongation factor EF-TU for hydrophobic peptides. Biochemical and Biophysical
Research Communications, 296, 749-754.
Manten A., Van Wijngaarden L.J. (1969) Development of drug resistance to rifampicin.
Chemotherapy, 14, 93–100.
Mariam D.H., Mengistu Y., Hoffner S.E. and Andersson D.I. (2004) Effect of rpoB mutations
conferring rifampin resistance on fitness of Mycobacterium tuberculosis. Antimicrobial
Agents and Chemotherapy, 48, 1289-1294.
Merle C., Faure D., Urdaci M-C and Le Henaff C. (2003) Purification and characterization of a
membrane glycoprotein from the fish pathogen Flavobacterium psychrophilum. Journal
of Applied Microbiology 94, 1120–1127.
Mesa, M.G., Maule, A.G. & Schreck, C.B. (2000) Interaction of infection with Renibacterium
salmoninarum and physical stress in juvenile chinook salmon: Physiological responses,
disease progression, and mortality. Transactions of the American Fisheries Society, 129,
158-173.
Moller J.D., Ellis A.E., Barnes A.C. and Dalsgaard I. (2005) Iron acquisition mechanisms of
Flavobacterium psychrophilum. Journal of Fish Diseases 7, 391-398.
Moorman D.R. and Mandell G.L. (1981) Characteristics of rifampin-resistant variants obtained
from clinical isolates of Staphylococcus aureus. Antimicrobial Agents and
Chemotherapy, 20, 709-713.
Olczak T., Sroka A., Potempa J. and Olczak M. (2008) Porphyromonas gingivalis HmuY and
HmuR: further characterization of a novel mechanism of heme utilization. Archives of
Microbiology 189, 197–210.
Olczak T., Wojtowicz H., Ciuraszkiewicz J. and Olczak M. (2010) Species specificity, surface
exposure, protein expression, immunogenicity, and participation in biofilm formation of
Porphyromonas gingivalis HmuY. BMC Microbiology 4: 134-144.
Plant K.P., LaPatra S.E and Cain K.D. (2009) Vaccination of rainbow trout, Oncorhynchus
mykiss (Walbaum), with recombinant and DNA vaccines produced to Flavobacterium
psychrophilum heat shock proteins 60 and 70. Journal of Fish Diseases 32, 521-534.
Plant K.P., LaPatra S.E., Call D.R. and Cain K.D. (2011) Immunization of rainbow trout,
Oncorhynchus mykiss (Walbaum), with Flavobacterium psychrophilum proteins
elongation factor-Tu, SufB Fe-S assembly protein and ATP synthaseβ. Journal of Fish
Diseases 34, 247–250.
22
Shah D.H., Cain K.D., Wiens G.D. and Call D.R. (2008) Challenges associated with
heterologous expression of Flavobacterium psychrophilum proteins in Escherichia coli.
Marine Biotechnology, 10, 719-730.
Silverstein, J.T., Vallejo, R.L., Palti, Y., Leeds, T.D., Rexroad, C.E., Welch, T.J., Wiens, G.D. &
Ducrocq, V. (2009) Rainbow trout resistance to bacterial cold-water disease is
moderately heritable and is not adversely correlated with growth. Journal of animal
science, 87, 860.
Stoller G., Rucknagel K.P., Nierhaus K.H., Schmid F.X., Fischer G. and Rahfeld J-U. (1995) A
ribosome-associated peptidyl-prolyl cis/trans isomerase identified as the trigger factor.
EMBO Journal, 14, 4939-4948.
Taylor, P.W. (2004) Detection of Flavobacterium psychrophilum in eggs and sexual fluids of
Pacific salmonids by a polymerase chain reaction assay: Implications for vertical
transmission of bacterial coldwater disease. Journal of Aquatic Animal Health, 16, 104-
108.
Vatsos, I.N., Thompson, K.D. & Adams, A. (2002) Development of an immunofluorescent
antibody technique (IFAT) and in situ hybridization to detect Flavobacterium
psychrophilum in water samples. Aquaculture Research, 33, 1087-1090.
Wilson D.N. and Nierhaus K.H. (2005) Ribosomal proteins in the spotlight. Critical Reviews in
Biochemistry and Molecular Biology, 40, 243-267.
Wojtowicz H., Guevara T., Tallant C., Olczak M., Sroka A., Potempa J., Sola M., Olczak T. and
Gomis-Rüth F.X. (2009) Unique structure and stability of HmuY, a novel heme-binding
protein of Porphyromonas gingivalis. PLoS Pathogens, e1000419, 1-11.