Coldwater disease prevention and control through vaccine development and

diagnostic improvements

Investigators: Ken Cain1, Doug Call2, Scott LaPatra3, Gary Fornshell4

Industry partner/advisor: Jim Parsons5

Other partners: Greg Wiens (USDA)6

(1)  Department of Fish and Wildlife Resources and Aquaculture Research Institute, University of Idaho, Moscow ID 83844-1136. Email: kcain@uidaho.edu

(2)  Dept. Vet. Micro. & Path., Washington State University, Pullman, WA 99164-7040. Email: drcall@wsu.edu

(3)  Clear Springs Foods, Inc. Research Division, PO Box 712, Buhl, ID 83316 (4)  University of Idaho, Twin Falls County Extension, Twin Falls, ID 83301 (5)  Troutlodge, Inc. Sumner, WA 98390 (6)  USDA, National Center for Cool and Coldwater Aquaculture, Kearneysville, WV

25430

Project Rationale (WRAC)

“Two Pronged” approach to prevention

and/or control of CWD

1. Vaccine development – improve antigen selection

2. Disease management •  Diagnostic improvements (validation

of broodstock culling program)

1. Identify potential vaccine candidates using in vivo-induced antigen technology (IVIAT)

•  Produce and test recombinant proteins 2. Validate quantitative diagnostic assays (ELISA,

ovarian fluid filtration FAT) •  Correlate assay results to risk of vertical

transmission or disease susceptibility in progeny •  Establish threshold levels for broodstock/egg culling

3. Based on results from objective 2 •  Develop other assays (e.g. real-time qPCR) for

quantification of infection (ovarian fluid) 4. Develop outreach/extension products

Project Objectives

Objective 1: Identify potential vaccine candidates using in vivo-induced antigen technology

pET30vector

pET30

Culture F. psychrophilum

Extract genomic DNA

Fragment DNA

Clone DNA fragments and insert into expression vector

Transform vectors into expression competent E. coli

Array clones and induce expression

Pool serum from trout that have recovered from BCWD

Absorb antisera against cells grown in broth culture

Antisera with antibodies specific for antigens expressed in vivo

Transfer clones to nitrocellulose membrane

Retrieve immuno-reactive clones for sequencing and characterization

pET30vectorpET30vector

pET30pET30

Culture F. psychrophilum

Extract genomic DNA

Fragment DNA

Clone DNA fragments and insert into expression vector

Transform vectors into expression competent E. coli

Array clones and induce expression

Pool serum from trout that have recovered from BCWD

Absorb antisera against cells grown in broth culture

Antisera with antibodies specific for antigens expressed in vivo

Transfer clones to nitrocellulose membrane

Retrieve immuno-reactive clones for sequencing and characterization

Attenuated vaccine approach is feasible

CSF259-93.B17 is attenuated

CSF259-93.B17 conveys significant protection against experimental challenge

250kDa

2001501007550

37251510

pH 3 pH 10

250

kDa

200150100

7550

37251510

pH 3 pH 10

Fig. 3. Two dimensional PAGE experiments for CSF 259-93 (wild-type; top panel) and B.17 (attenuated; bottom panel) strains. Dark ovals highlight protein spots that are clearly differentially regulated. Lighter ovals represent protein spots or regions where additional resolution is needed to ascertain differences.

250kDa

2001501007550

37251510

pH 3 pH 10

250

kDa

200150100

7550

37251510

pH 3 pH 10

250kDa

2001501007550

37251510

pH 3 pH 10

250

kDa

200150100

7550

37251510

pH 3 pH 10

Fig. 3. Two dimensional PAGE experiments for CSF 259-93 (wild-type; top panel) and B.17 (attenuated; bottom panel) strains. Dark ovals highlight protein spots that are clearly differentially regulated. Lighter ovals represent protein spots or regions where additional resolution is needed to ascertain differences.

Both wild-type and attenuated strains have unique expression patterns for >8 proteins

Figure 4. Preliminary results showing protection against F. psychrophilum challenge after immunization with a mixture of four different recombinant proteins that were expressed in V. parahaemolyticus and prepared as crude lysates (2nd column, P=0.05). Immunization with column purified proteins and Freund’s complete adjuvant (FCA) produced significantly lower survivorship.

Figure 4. Preliminary results showing protection against F. psychrophilum challenge after immunization with a mixture of four different recombinant proteins that were expressed in V. parahaemolyticus and prepared as crude lysates (2nd column, P=0.05). Immunization with column purified proteins and Freund’s complete adjuvant (FCA) produced significantly lower survivorship.

•  Lysate screening is feasible

•  Mixed protein format feasible

•  V. parahaemolyticus may be an effective adjuvant for aquaculture applications

Improved expression system once antigens identified

Moving forward 1.  Halt IVIAT optimization efforts

•  Move directly to protein testing based on B.17 and CSF259-93 proteomics

•  Identify/express antigens/genes of interest

2.  Screen using mixed lysate strategy

3.  Determine if Vibrio lysate can be used for immersion delivery

Progress (assays using FL-43): –  ELISA optimization

•  Evaluating changes in ELISA O.D. values over the course of a bacterial challenge

–  Validated MF-FAT using ovarian fluid samples from two different hatcheries

–  Developed a smear FAT and indirect FAT that can be used with a variety of tissues

Objective 2: Validate quantitative diagnostic assays (ELISA, ovarian fluid filtration FAT)

MF-FAT Validation

‒  118 ovarian fluid samples were collected in Spring 2009 from steelhead returning to Dworshak National Fish Hatchery (Orofino, ID) and rainbow trout broodstock at J. Perry Egan Hatchery (Bicknell, UT)

–  Samples were transported to UI on either the day of collection (Dworshak) or within 48 hours of collection (Egan)

–  Samples were tested for F. psychrophilum by nested PCR, culture techniques, and MF-FAT

Results

DNFH Egan

# % # %

Total samples 99 - 19 -

YPB growth 31 31 0 0

MF-FAT 29 29 9 47

‒ 38 of 118 samples analyzed by MF-FAT were positive for F. psychrophilum ‒ MF-FAT is sensitive

enough to detect F. psychrophilum when culture techniques alone cannot

Controlled challenge experiment

•  Assess changes in ELISA optical density values by sampling kidneys from fish injected with PBS or F. psychrophilum

•  Three treatments – Mock-infected: PBS – High: 107 CFU ml-1 – Low: 105 CFU ml-1

•  Triplicate groups per treatment – 25 juvenile rainbow trout per group

Challenge Sampling Design

•  Prior to challenge, 5 fish were randomly sampled

•  Random samples were taken every other day for two weeks and then once a week for the remaining two weeks – Kidney, liver, and spleen were streaked on TYES

plates and monitored for YPB growth – Kidney and spleen imprints were made for IFAT – Kidney was extracted from all fish and pooled for

each treatment to be used in the ELISA •  20% of the daily mortalities sampled using

the same protocols

Challenge Results

•  Bacteria – Bacterial levels in kidney homogenate ranged

from 103 to 106 CFU ml-1 as determined by plate counts

•  FAT – Preliminary results indicate that this method is

able to discriminate between infected and uninfected fish as determined by culture of kidney, liver, and spleen

•  ELISA – Underway

Upcoming work

•  Samples (kidney and ovarian fluid) will be collected from Troutlodge in November to determine assay yields in relation to broodstock infection levels

•  Experiments assessing vertical transmission in progeny grouped according to broodstock infection levels will start in February 2010

•  Broodstock samples will also be obtained from Clear Springs to establish infection rankings –  Additional in vivo trials following progeny may be

implemented

Objective 3: Development of a qPCR assay

– Testing will begin using published qPCR approach

– New probe(s) will be designed to the gene encoding the antigen that is recognized by MAb FL43

•  Putative sequence for this gene was recently resolved by the Call Lab at WSU

•  Additional studies are being done to confirm immunoreactive to MAb FL43

– Once sequence has been verified, assay conditions, probe specificity, and detection limit will be determined

– Tissue extraction procedures will also be optimized

Objective 4: Develop outreach/extension products

– General article (Waterlines)

– Presentations at meetings (Park City –AFS/FHS)

– Partnered (licensing agreement) with ImmunoPrecise Antibodies, Inc. to produce and commercialize Mab FL-43

•  Monoclonal now available and company will further develop assay kits based on results from this study.

Coldwater disease prevention and control through vaccine development and diagnostic improvements

Year 1 Year 2

Year 3

Year 4

Project leader(s)

Objective 1: Shift antigen selection strategy to proteomic/genomic comparison of B.17 and Wild type 259-93 strain

XXXX XXXX Call

Select vaccine candidates XXXX Call/Cain

Produce recombinant protein XXXX XX Call/Cain

Test recombinant vaccine candidates XXXXX XX LaPatra/Cain

Objective 2: Validate and further develop quantitative and simple diagnostic assays (ELISA, Ovarian fluid filtration FAT)

XXXX XXXXX Cain

Determine biological significance of infection levels in relation to vertical transmission and/or susceptibility

XX XXXXX Cain

Establish threshold levels for culling broodstock and/or eggs

XXX X Cain/Call

Objective 3: Develop a quantitative PCR assay XX XXXXX Cain/Call

Objective 4: Develop outreach/extension products XX XXXXX XXXX Fornshell

Submit manuscripts for peer-reviewed publication XXX XXXXX Cain/Call

Schedule for completion of objectives

LaFrentz, B.R., LaPatra, S.E., Call, D.R., Wiens, G.D. and Cain, K.D. Proteomic analysis of Flavobacterium psychrophilum cultured in vivo and in iron-limited media. Diseases of Aquatic Organisms (In Press) Plant, K.P., LaPatra, S.E., and Cain, K.D. 2009. Vaccination of rainbow trout (Oncorhynchus mykiss) with recombinant and DNA vaccines produced to Flavobacterium psychrophilum heat shock proteins 60 and 70. Journal of Fish Diseases 32(6): p. 521-34 Lindstrom, N.M., Call, D.R., House, M.L., Moffitt, C.M., and Cain, K.D. 2009. A quantitative enzyme-linked immunosorbent assay (ELISA) and filtration-based fluorescent antibody test (FAT) as potential tools to screen broodstock for Flavobacterium psychrophilum infection. Journal of Aquatic Animal Health 21(1): p. 43-56 Cain, K.D. 2009. Strategies for Control and Prevention of Coldwater Disease. Waterlines newsletter 15 (1): p. 18-20 Long, A., Call, D.R., and Cain, K.D. 2009. Comparison of diagnostic techniques for detection of Flavobacterium psychrophilum in ovarian fluid. Talk presented at the 50th Western Fish Disease Workshop and AFS Fish Health Section Annual Meeting. Park City, Utah. June 7-10.

Related Publications/Presentations

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