Progress 06/01/14 to 01/31/16
Outputs
Target Audience:We now have active research collaborations with 5 groups that do either shellfish research and/or commercial production of larvae. Three are in the US (Gary Richards, USDA, ARS, Dover, DE; Chris Langdon, Hatfield Marine Science Center, Newport, OR; and Lauren Gregg, Virginia Institute of Marine Science, Gloucester Point, VA), one in New Zealand (Sarah Cumming, SpatNZ, Nelson, NZ) and one in Australia (Wayne O'Connor, Port Stephens Fisheries Institute, Taylors Beach, AU). We have also interacted with many commercial shellfish hatchery managers through email and personal contact at research conferences. In particular, we have had multiple visits and phone calls with hatchery managers at three Pacific Northwest hatcheries (Dr. Benoit Eudeline at Taylor Shellfish, Chris Jones at Coast Seafoods, and Sue Cudd at Whiskey Creek). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two post-doctoral researchers (Carla Schubiger and Aimee Reed at OSU), a Research Associate (Yan Campbell at mAbDx) and two undergraduate research assistants (Eric Hobson at OSU and Fintan Doyle at mAbDx) were trained in immunoassay development. In addition, both PIs visited Pacific Northwest shellfish hatcheries had extensive discussions with hatchery operators while viewing hatchery operations. These visits have provided extremely relevant practical experience and better understanding of the problems facing hatchery operators and insights into how best to deploy the Vibriosis RapidTest effectively in the field. In a recent NSF funded CNIC project, Co-PI Claudia Häse explored the exciting possibility of using the Vibriosis RapidTest to detect coral pathogens in Australia. Coral disease has emerged as a significant threat to coral reef ecosystems, with dramatic declines in coral cover and diversity of reefs worldwide. Dr. Häse worked closed with Dr. David Bourne at the Australian Institute for Marine Science, met with many Australian Aquaculture scientists and introduced them to the Vibriosis RapidTest. How have the results been disseminated to communities of interest?The PIs have interacted with shellfish hatchery managers through visits, phone calls and personal contacts at conferences. In addition, much of the progress described above has been presented to the research and shellfish aquaculture community at scientific conferences through presentations and written reports as described in the "Products" and "Other Products" sections. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Specific Objective #1. We successfully transitioned a crude prototype device into a mature, highly sensitive yet simple diagnostic test. The "Vibriosis RapidTest" is a true diagnostic Lateral Flow Assay (LFA) that is robust (rugged, durable and reliable), extremely easy to use (simply add a few drops of unprocessed seawater to the sample port), rapid and highly sensitive (Phase I sensitivity goals were met and exceeded, see Specific Objective #2). The composition and performance of each critical component in the device was assessed and then modified and optimized through a series of iterative 'generations' of the device, culminating in a cassette-enclosed easy-to-use product. Preparation and deployment of the paired critical immunocapture and immunodetction reagents was improved and standardized to improve both the sensitivity and reproducibility of the assay. For example, whereas prototype tests were produced by manually applying spots of capture reagents in approximate zones on partially completed dipsticks and liquid (perishable) detection reagents had to be mixed individually with each sample immediately before each test, high quality robotics are now used to apply immunocapture and detection reagents precisely in discrete linear zones on large production cards and a semi-automated cutting instrument is used to process the cards into individual dipsticks. Individual dipsticks are then mounted and sealed in durable plastic cassettes that have a sample port and a visual readout window over the capture line zones. The finished cassettes have a shelf life (stored simply at room temperature) of at least 8 months and test samples can be added directly to the sample port without any processing or mixing of reagents. Specific Objective #2. The sensitivity goal has been met and exceeded. The limit of detection (LOD) of purified recombinant VcpA by the current Vibriosis RapidTest is 0.005 μg/ml (5 ng/ml), which is 20-fold below the concentration of VcpA that causes mortality of oyster larva under laboratory conditions (0.1 μg/ml). This represents a 100-fold increase in sensitivity over the LOD (500 ng/ml) of the prototype test. We have also corrected the identities of the antigen (VcpA) and bacterial pathogen (Vibrio coralliilyticus) targeted by the Vibriosis RapidTest. Until recently many strains of V. coralliilyticus, including pathogenic strains that impact Pacific Northwest shellfish hatcheries, were mis-identified as Vibrio tubiashii in research literature. Specific Objective #3. A visual reference card has been successfully prepared. The card displays images of a panel of test cassettes loaded with samples containing increasing concentrations of recombinant VcpA diluted in seawater. The visual reference card can be used to estimate the concentration of VcpA in unknown samples over a wide range of VcpA concentrations (5 - 320 ng/ml) which importantly spans the concentration of VcpA that is toxic to larvae in laboratory experiments. As the concentration of VcpA increases in a sample, the "Test" line becomes increasing darker allowing rapid visual estimation of the concentration of VcpA in test samples. We are now working with several research shellfish hatcheries (below) to determine "real-world" action levels of VcpA at which production runs are at significant risk of being compromised by toxic vibriosis. Specific Objective #4. In an exciting development, we and collaborators recently used the Vibriosis RapidTest to demonstrate for the first time V coralliilyticus-associated mortalities of larval oysters in a shellfish hatchery. Importantly, test results with the Vibriosis RapidTest were in complete agreement with testing done in parallel using gold-standard PCR-based assays for V coralliilyticus. A manuscript describing this work has been submitted for publication (Gary Richards, USDA-ARS, personal communication). A caveat to this initial successful hatchery study is that sample collection and testing involved overnight culture of hatchery samples. Therefore, we are now working with collaborating hatcheries to establish improved sample collection and testing conditions that will generate same-day results and allow operators to take same-day actions in response to V coralliilyticus contamination detected with the Vibriosis RapidTest.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
A rapid (1 hour), simple test for the pacific oyster pathogen Vibrio coralliilyticus based on detection of the shellfish vibriosis toxin VcpA with a sensitive lateral flow dipstick immunoassay, Marusich, M.F., Schubiger, C. and Hase, C. An invited oral presentation delivered by project PI Marusich at the 2016 World Aquaculture Society Annual Meeting, 2/22/16-2/26/16.
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Progress 06/01/14 to 05/31/15
Outputs
Target Audience:The PIs have had ongoing interactions, including visits and phone calls with the hatchery managers at three regional hatcheries, namely Dr. Benoit Eudeline at Taylor Shellfish, Chris Jones at Coast Seafoods, and Sue Cudd at Whiskey Creek. In addition, we presented details of the Vibriosis RapidTest technology and initial results to many other members of the shellfish aquaculture and research community at the 107th National Shellfisheries Association Annual Meeting, 3/22/15-3/26/15. A poster titled: "Development of a lateral flow immunoassay to detect VcpA metalloprotease of the Pacific oyster pathogen Vibrio coralliilyticus", was presented at this conference by Reed, Hobson, Marusich and Häse. Collaborator Eric Hobson presented a report entitled "Lateral Flow Immunoassay for the detection of Vibrio Coralliilyticus zinc-metalloprotease VcpA" to the Pathogen Research in Progress group at Oregon State University. Finally, a manuscript is being prepared for submission that describes using the current version of our novel rapid test for vibriosis (the Vibriosis RapidTest) to demonstrate for the first time Vibrio coralliilyticus-induced mortalities of larval oysters in a shellfish hatchery. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have trained two post-doctoral researchers (Dr. Carla Schubiger and Dr. Aimee Reed at OSU) and a Research Associate (Dr. Yan Campbell at mAbDx) and two undergraduate research assistants (Eric Hobson at OSU and Fintan Doyle at mAbDx) in immunoassay development and specifically, in aspects of lateral flow immunoassay technology. In addition, both Principle Investigators have had the opportunity to visit a number of Pacific Northwest shellfish hatcheries, engage in extensive discussions with hatchery operators while viewing on-going operations, conduct and oversee sampling for research analysis. These visits and interactions have provided extremely relevant practical experience and better understanding of the vibriosis problems facing hatchery operators and insights into possible solutions - in particular how best to deploy the Vibriosis RapidTest most effectively in real-world conditions. Finally, in a recent NSF funded CNIC project, Co-PI Claudia Häse explored the exciting possibility of using our Vibriosis RapidTest to detect coral pathogens. Coral disease has emerged as a significant threat to coral reef ecosystems, with dramatic declines in coral cover and diversity of reefs worldwide. During her visit, Dr. Häse worked closed with Dr. David Bourne at the Australian Institute for Marine Science (AIMS) and had the opportunity to meet with many Australian Aquaculture scientists, learn from them and also introduce them to the potential of our rapid LFA technology. How have the results been disseminated to communities of interest?Some of the results described here have already been presented to the research and shellfish aquaculture community in the form of a poster presentation and discussions at the recent 107th National Shellfisheries Association Annual Meeting, 3/22/15-3/26/15. A poster titled: "Development of a lateral flow immunoassay to detect VcpA metalloprotease of the Pacific oyster pathogen Vibrio coralliilyticus", was presented by Reed, Hobson, Marusich and Häse. A manuscript is being prepared for submission that describes using the current version of our novel rapid test for vibriosis (the Vibriosis RapidTest) to demonstrate for the first time Vibrio coralliilyticus-induced mortalities of larval oysters in a shellfish hatchery. Collaborator Eric Hobson presented a report entitled "Lateral Flow Immunoassay for the detection of Vibrio Coralliilyticus zinc-metalloprotease VcpA" to the Pathogen Research in Progress group at Oregon State University. The PIs have also had ongoing interactions, including visits and phone calls with regional shellfish hatchery managers. Finally, the Vibriosis RapidTest and our work to help Pacific Northwest shellfish hatcheries monitor and mitigate the adverse impacts of vibriosis has also been the subject of an informative research news release from the office of U.S. House of Representatives Congressman Peter DeFazio (4th District of Oregon, the site of mAbDx, Inc., the Small Business Concern): http://defazio.house.gov/media-center/press-releases/defazio-applauds-federal-grant-for-eugene-innovator, using information provided by the Technology Transfer Offices of Oregon State University (the site of Co-Project Director Claudia Häse) and the University of Oregon (the Small Business Concern mAbDx, Inc., is a spinout from the University of Oregon). What do you plan to do during the next reporting period to accomplish the goals?Ongoing work is focused on two issues. First, to document performance of the visual reference card for rapid, quantitative measurements of VcpA in seawater made using the current "Type 4B1" cassette-mounted dipsticks. And second, to continue working with collaborating shellfish hatcheries to determine the optimal hatchery sample collection and testing conditions that generate same-day results that would allow operators to take same-day actions in response to detected contamination. The visual reference card contains images of a series of dipsticks processed with samples that contain increasing, known concentrations of VcpA. In effect, the reference card is a visual representation of a standard curve of VcpA in seawater. Using such cards as guides, observers can determine where in the standard curve an unknown test sample's dipstick signal fits, and thereby quickly and accurately measure the concentration of VcpA in the sample. We have determined that the effective visual working range for the current Type 4B1 Vibriosis RapidTest is 0.005 - 0.5 μg of recombinant VcpA per ml seawater. Therefore, visual reference cards will be made using this range and the accuracy of visual readout determined by running 4 samples at different VcpA concentrations and having 8 naïve volunteers make visual readouts of the resulting dipsticks. If necessary, the reference card will be optimized by adjusting the "spacing", i.e., concentrations displayed, of the standard curve. The goal is to attain intra- and inter- assay accuracy better than 15% throughout the critical mid-range of 0.01 - 0.2 µg/ml of VcpA because previous work has established that VcpA causes measureable larval toxicity in laboratory cultures at concentrations of 0.1 µg/ml and below. Therefore, 0.1 μg/ml VcpA may be a red-line action level for hatchery operators. The visual reference card will be useful in subsequent hatchery studies in Phase II to determine if 0.1 µg/ml VcpA is indeed a useful real-world action level and if not, to define an appropriate action level. We will also continue ongoing work at three Pacific Northwest shellfish hatcheries to determine optimal hatchery sample collection and testing conditions for VcpA dipstick testing. We plan to test samples taken from various time and processing points in the larval development pipeline, and to focus on production runs that exhibit unexplained retarded growth. We will also explore options to increase sensitivity of sampling by focusing analysis on stunted larvae (obtained routinely in hatcheries by sieving and sorting larval subpopulations during larval subculture and expansion steps) and by concentrating the sorted larvae for short times (30-60 min) in seawater, to allow maximal expression and release of VcpA. Our recent analysis of laboratory cultures of larvae infected with Vibrio coralliilyticus suggests that secreted levels of VcpA peak after 30-60 minutes of culture time. Therefore, this brief concentrated culture step may provide a significant increase in test sensitivity using hatchery samples.
Impacts
What was accomplished under these goals?
Specific Objective #1. We have successfully transitioned a prototype device into a completely self-contained, self-developing and highly sensitive diagnostic test. The test, now named the "Vibriosis RapidTest" is now a true diagnostic Lateral Flow Assay (LFA) that is robust (rugged, durable and reliable), extremely easy to use (simply add a few drops of unprocessed seawater to the sample port), rapid and highly sensitive (the Phase I sensitivity performance goal has also been met and exceeded, see Specific Objective #2 below). The performance of each critical kit component in the device was assessed and then modified and optimized as needed through a series of iterative 'generations' of the device, culminating in a complete cassette-enclosed easy-to-use device. Application and immobilization of immunocapture reagents in the "Test" line and "Control" line zones was improved and standardized to improve both the sensitivity and reproducibility of the assay. For example, whereas the prototype tests were produced by manually applying spots of capture reagents in approximate zones on partially completed dipsticks, high quality robotics are now used to apply the immunocapture reagents precisely in defined linear zones on large production cards and an automated cutting instrument is used to process the cards into individual dipsticks. Individual dipsticks are then sealed in durable plastic cassettes that have a sample port and a visual readout window over the capture line zones. Specific Objective #2. NOTE: Until recently many strains of V. coralliilyticus, including pathogenic strains that impact pacific North West shellfish hatcheries, were mis-identified as Vibrio tubiashii in research literature. Therefore, the shellfish toxin previously known as VtpA has been re-named VcpA. The sensitivity goal has been met and exceeded. The limit of detection of purified recombinant VcpA by the current (6th generation) Vibriosis RapidTest is 0.005 µg/ml (5 ng/ml), which is 20-fold below the concentration of VcpA that causes mortality of oyster larva under laboratory conditions (0.1 µg/ml). This represents a 100-fold increase in sensitivity over the previous limit of detection of 0.5 µg/ml VcpA using the pre-Phase I prototype Vibriosis RapidTest. First, we adapted and brought in-house a custom protocol to manufacture High Density Ellipsoid (HDE) gold nanoparticles to use as the assay's detector component. Custom made HDE gold nanoparticles proved to be more sensitive detectors than the commercially available gold nanoparticles used in the prototype Vibriosis Rapid Test. An additional benefit of making HDE gold nanoparticles in-house is that the cost of these key materials is now greatly reduced (more than 10-fold) and both their supply and quality can be assured internally. Next, orientation of the key immunologic components, namely the paired "capture" and "detector" monoclonal antibodies (mAbs), was reversed completely after it was determined that the new detector mAb conjugated to HDE gold nanoparticles was both much more stable and had superior performance than did the previous gold-mAb detector conjugate. The new orientation of capture/detector mAbs generates extremely strong signals when positive control samples are tested, but generates no detectable background signal when blank control samples are tested and scored between 20 minutes and 4 hours after loading. This modification therefore resulted in both a greatly improved limit of detection (LOD) and an expanded dynamic range. Performance and long-term stability of the gold-detector mAb conjugate was optimized further by reviewing and modifying the conjugation protocol, including reaction pH, reactant concentrations, blocking conditions and incorporation of additional stabilizers. Importantly, these changes also further reduced the materials cost per test as significantly less detector mAb is now needed. We then evaluated immobilization of the new capture mAb on various forms of lateral flow nitrocellulose to find the best matrix for immobilization and mAb stability. Overall test performance was then optimized by producing a wide variety of dipsticks using multiple permutations of commercially available LFA components and assay matrix materials, including various types of nitrocellulose and conjugate release pads, each having different protein binding and flow rate characteristics, and absorbent pads with different capacities. A variety of different modes to apply easily released gold-mAb detector conjugate and blocking agents were also tested. Finally, performance of each dipstick component permutation was evaluated and the optimal combination of components selected on the basis of achieving: 1) extremely low (essentially undetectable) background when blank samples are run and read between 20 min and 4 hr after loading, 2) high sensitivity (low Limit of Detection) to detect VcpA-containing samples, and 3) rapid flow to ensure readability of the assay within ~20 minutes (maximum sensitivity is generated within 2 hours and background from null samples is negative for at least 4 hours). A low protein binding sample filtration pad was incorporated immediately under the sample addition well to facilitate use of turbid samples without the need for processing or clarification. Finally, we evaluated a wide range of commercially available plastic cassette housings and selected the one that facilitated rapid signal development and optimal self-washing capabilities. Specific Objective #3. Standard curves suitable for the visual reference card have been generated using defined concentrations of recombinant VcpA diluted in seawater. Increasing concentrations of VcpA generate increasingly dark Test lines. We are now finishing work to determine the optimal concentration steps for reference samples that will facilitate rapid, accurate visual readout of the concentration of VcpA in test samples. We will then evaluate the accuracy and reproducibility of reference card aided visual readout of test samples containing various concentrations of VcpA. Specific Objective #4. In an exciting development, we and collaborators recently used theVibriosis RapidTestto demonstrate for the first timeVibrio coralliilyticus-induced mortalities of larval oysters in a shellfish hatchery.Importantly, test results with theVibriosis RapidTestwere in complete agreement with testing done in parallel using gold-standard PCR-based assays forVibrio coralliilyticus.A manuscript describing this work is being completed and will be submitted for publication soon.After the manucript is accepted for publication, details of the study will be included both in the Final Report for Phase I work and in our upcoming application for Phase II support to continue this promising line of work.One caveat to this initial hatchery study is that sample collection and testing involved overnight culture of hatchery samples. Therefore, we are now working with several collaborating hatcheries to determine sample collection and testing conditions that generate same-day results and allow operators to take same-day actions in response to detected contamination.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Development of a lateral flow immunoassay to detect VcpA metalloprotease of the Pacific oyster pathogen Vibrio coralliilyticus , Reed, A., Hobson, E., Marusich, M.F. and H�se, C. Poster presentation at the 107th National Shellfisheries Association Annual Meeting, 2015.
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