Recipient Organization
GASKIYA DIAGNOSTICS, INC.
8 MARKET PL STE 300
BALTIMORE,MD 212024113
Performing Department
(N/A)
Non Technical Summary
Aquaculture is an integral and critical part of feeding the world's burgeoning population. Aquatic diseases cost the industry $6 billion each year and threaten global food security as well as the livelihoods of individual farmers. Typically, farmers are unaware of a disease issue until there are signs of advanced disease progression and/or high rates of mortalities. Timely diagnostic information is key to controlling outbreaks and mitigating the impact of disease. Testing of samples using typical laboratory methods rarely results in actionable information as the delay between sampling and results can be on the order of days-to-weeks. Clearly, better technologies and methodologies are needed to address this unmet market need.Rapid, low-cost, on-site diagnostics are a necessary part of a comprehensive approach leading to disease mitigation and improvement of overall stock health. Through our customer discovery efforts, we have focused the current proposal to address the unmet need of cost-effectively detecting Infectious Hematopoietic Necrosis Virus in rainbow trout (Oncorhynchus mykiss) production facilities. Infectious Hematopoietic Necrosis Virus (IHNV), the cause of Infectious Hematopoietic Necrosis (IHN), is a chronic disease of Salmonid fish, a family which includes rainbow trout. Transmission between fish typically occurs through the water and contaminated feed, and the mortality rate is 60-75%. There is no available treatment or cure for the disease, so early detection and implementation of measures to control the spread are essential. We have developed a diagnostic platform technology that implements bioengineered proteins to detect disease targets. The capture proteins are incorporated into low-cost, user-friendly tests that can be performed on-site by farmers of any skillset and require no expertise or additional equipment to perform or interpret results. We previously demonstrated proof-of-concept with a paper-based immunoassay coupled to polymerization-based amplification for the detection of protein-based biomarkers for HRP2, the primary protein biomarker of malaria, with sensitivity as low as 70pM. In the first stage of IHNV test development, IHNV-specific bioengineered proteins will be identified, isolated, and validated. In the second stage of IHNV test development, the paper-based test assembly will be optimized as part of an effort to understand the biological and functionally meaningful limits of the developed assay. The resulting prototype will rapidly and selectively bind the IHNV target and yield stable, easily interpretable, colorimetric results. Our initial product version will be further developed and improved upon through market trials and close collaboration with our customers to ready our system for commercialization.U.S. trout production is concentrated into 16 states, with Idaho as the largest producer with 40% of the total value of fish sold. Disease is a constant worry on farms, with upwards of $30 million lost to disease every year, predominantly due to deaths in fry in hatch houses. Per our conversations, many trout operations ship out samples to centralized labs for disease analysis, however the time-to-results is in weeks due to the shipping and analysis time constraints. For farms that currently send out samples for testing, we believe that our tests will be used in conjunction with the practice of sending samples to outside labs or potentially supplant some of this existing market based on time-to-results, cost, and ease of use. Some of the larger and more well-funded farms have already resorted to setting up their own labs with trained personnel on site, demonstrating that operators are willing to invest significant capital to decrease the time-to-results. Even the sophisticated farmers with on-site labs have indicated an interest in incorporating rapid testing methods into their workflow. The technological developments proposed are poised to enhance the production efficiency of aquaculture (diseased fish grow slower and/or die) by providing a cost-effective, unambiguous, and user-friendly solution to monitoring the health of against common diseases. Additionally, the development of our enabling technology de-risks the cultivation process, lowering the barrier to entry. More specifically, understanding the qualitative (presence/absence) and quantitative (how much) nature of IHNV, provides actionable information that could result in the increased domestic production of trout. This further manifests as increased production efficiency, increased revenue from exports, and creation of new jobs. Providing U.S.-based farmers/distributers with a competitive advantage can also help to attenuate the current $16.8 billion seafood trade deficit.
Animal Health Component
75%
Research Effort Categories
Basic
0%
Applied
75%
Developmental
25%
Goals / Objectives
GoalsRegardless of species, region, or type of farm operation, disease is the single largest cause of economic loss in aquaculture. When disease hits a farm, the results are often catastrophic. Rainbow trout (Oncorhynchus mykiss) is one of the most heavily farmed species in U.S.-based aquaculture, so when a crop fails due to disease, the impact reverberates throughout the supply chain. To mitigate the risk of disease, we propose the development of a pond-side diagnostic biosensor platform for the rapid detection of pathogens common in rainbow trout (O. mykiss) aquaculture. Our paper-based, thermostable, rapid diagnostic test requires no training or expensive/specialized equipment and provides an easily interpretable colorimetric result within minutes. Low cost, high sensitivity and accuracy, as well as rapid results facilitate increased monitoring and provide actionable information where it is needed the most. Mitigating the largest risk in O. mykiss aquaculture lowers the barrier to entry and provides U.S.-based aquaculture with the competitive edge that could result in increased domestic production, thereby reducing expenditures on imports, increasing revenue from exports, and potentially creating new jobs.We propose to apply our platform technology to the development of a low-cost, paper-based, easy-to-use diagnostic test against the common Salmonid virus, Infectious Hematopoietic Necrosis Virus (IHNV), which causes Infectious Hematopoietic Necrosis (IHN). As the disease is incurable, infected animals are culled as soon as they begin showing symptoms in an attempt prevent further spread of the disease; this is often too late as the clinical manifestation of the disease occurs once the disease has spread throughout the entire crop. Pondside diagnostics do exist in the form of semi-portable PCR machines and antibody-based tests, but both require trained personnel, are often cost prohibitive, and require additional equipment not found on most farms. A cost-effective, simple-to-use, and equipment-free prototype test for detecting IHNV will address a substantially unmet diagnostic need for O. mykiss farmers.ObjectivesMajor objectives of the proposed work include (1) Generation of an IHNV-binding rcSso7d protein; (1.1) Initial screening and refinement, (1.2) Affinity determination and strain selection, (1.3) Sequencing and stable expression and (2) Optimization of reagents for IHNV detection; (2.1) Range-finding for individual reagents, (2.2) Establish dynamic range for diagnostic prototype. The proposed 8-month timeline is sufficient to complete the scope of the work. The resulting prototype will undergo further technological refinements as well as evaluation for user-friendliness before commercialization.EffortsWe plan to collaborate with extension services to reach farmers who are interested in monitoring the health of their stock. They can advise our development in terms of what products are most needed as well as eventually provide valuable feedback on the user-friendliness of our tests. Our focus in on U.S. farmers, but IHNV in trout is a global issue. We can work with extension services as well as non-governmental organizations around the world to both learn from and educate farmers on disease monitoring as a tool to maintain overall stock health. As a downtown Baltimore business, we plan to reach out to local students seeking internships as well as interface with and inform the local community about scientific innovations happening in their city. We will collaborate with established local outreach organizations to facilitate these connections.
Project Methods
Methods The proposed work will generate a vertical flow assay capable of detecting IHNV utilizing rcSso7d capture proteins coupled to an eosin-Y-based polymerization-based amplification (PBA) system. This project is specifically designed to demonstrate the impact of assembly conditions (i.e. reagent combinations) on detection limits. From this information, we can extrapolate the optimal conditions to produce a robust and reliable signal that is also highly sensitive to the minimum levels of viral load without the need for special equipment or training. We previously demonstrated proof-of-concept with a paper-based immunoassay with polymerization-based amplification for detection of protein-based biomarkers for HRP2-the primary protein biomarker of malaria-with sensitivity as low as 70pM. In the first task of IHNV test development--Generation of an IHNV-binding rcSso7d protein--IHNV-specific thermostable rcSso7d DNA-binding proteins will be identified, isolated, and validated. In the second task of IHNV test development--Optimization of reagents for IHNV detection--assay sensitivity, reagent composition, and dynamic range in the paper-based format will be explored and calibrated to generate meaningful test results for INHV. The resulting prototype will rapidly and selectively bind the IHNV target and yield stable, easily interpretable, colorimetric results.Task 1: Generation of an IHNV-binding rcSso7d protein. (1) A combinatorial library of EBY100 Saccharomyces cerevisiae cells expressing reduced charge Sso7d proteins (rcSso7d) - representing ~1.4 x 109 unique rcSso7d clones - will be screened for IHNV-binding variants. (2) Serial magnetic bead sorting and fluorescence-activated cell sorting (FACS) of increasing stringencies will be used to select binders against an IHNV virus-like particle (VLP) target. (3) To ensure sufficient affinity for IHNV, isolated rcSso7d strains will undergo affinity determination via yeast-surface display titration of VLPs conjugated with AF647 [0-250nM] in the presence of a fixed number of cells. (4) The geometric mean fluorescent intensity (MFI) for each condition will be used to calculate the EC50 for each selected strain and the top performing strain will be sequenced, cloned, and stability transfected into E.coli. (5) Within the expression system, the selected rcSso7d variant will be engineered to express a N-terminal hexahistidine tag (his-tag) and cellulose binding domain (CBD) to enhance protein retention and analyte detection, respectively.Analysis, Interpretation, and Controls: Mean fluorescence intensity (MFI) will be quantified using established methods and the ImageJ software package. Throughout this process, appropriate control conditions such as: absence of soluble IHNV-VLP antigen, absence of rcSso7d binding protein, and/or lysate from untransformed cells will be used as negative controls to ensure internal reliability and reduce false-positives. Anti-Infectious Hematopoietic Necrosis Virus (IHNV) rabbit polyclonal antibody (pAB) conjugated to Eosin-Y will serve as a positive control for rcSso7d as an IHNV capture protein; both species will be diluted to ensure equimolar representation of binding moieties. Biological and technical replicates will be performed in triplicate to establish each result and variability.Task 2: Optimization of reagents for IHNV detection. (1) Following generation and purification, engineered IHNV-binding rcSso7d-CBD constructs will be deposited on test zones (defined by hydrophobic ink) on unmodified cellulose chromatography paper for assessment purposes. (2) The concentration of individual reagents (rcSso7d; Eosin-Y; soluble VLP) will be systematically varied (0-300nM) to establish detection limits and inform optimization efforts. (3) Samples will be contacted with 20μL of an aqueous detection solution [200mM poly(ethylene glycol) diacrylate, 100mM 1-vinyl-2-pyrrolidinone, 150mM triethanolamine, 1.6mM phenolphthalein, 0.02N hydrochloric acid, and eosin Y) and incubated under ambient conditions for 3 minutes prior to photoinitiation by an ampliPHOX reader (λmax = 522nm, 30mW cm-2) for 30 seconds; the presence of hydrogel will be visualized by adding 1.5μL of 0.5 M NaOH to the surface. (4) The resulting colorimetric signals will be imaged at various time-points (t0-360 minutes) to determine stability of the signal.Analysis, Interpretation, and Controls: Colorimetric intensity (ΔCIE), mean fluorescence intensity (MFI) will be quantified using established methods and the ImageJ software package.Limits of detection (LOD) will be calculated in two manners: 1) visual LoD; the concentration where all replicates show a visible colorimetric response, and 2) calculated LoD (Figure 5); the minimum concentration that gives an average colorimetric intensity that is higher than the average intensity from the negative surface by three times the standard deviation of the mean from the negative surface. As above, anti-IHNV polyclonal antibody will serve as a positive control for rcSso7d capture protein. Negative controls will consist of absence of soluble IHNV-VLP target antigen, absence of capture protein, and/or photoionization event.EffortsOn-site testing puts the power of diagnostic testing right into the hands of farmers. There are roughly 700 farms in the U.S. currently cultivating O. mykiss, and the introduction of a rapid, cost-effective diagnostic that gives real-time information about IHNV will help them to lessen disease impact. As we continue to develop the test for IHNV, we will work with farmers to demonstrate the product, ensure that the testing platform is optimally user-friendly, and make adjustments as needed.EvaluationKey milestones by which we will measure the success of the project include1) Generation of IHNV-binding rcSso7d protein. (1.1) Initial screening and refinement, (1.2) Affinity determination and strain selection, (1.3) Sequencing and stable expression2) Optimization of reagents for IHNV detection. (2.1) Range-finding for individual reagents, (2.2) Establish dynamic range for diagnostic prototypeWe intend to work closely with trout farmers during all stages of development and beyond to ensure that we are developing a test that best meets their needs in terms of a sufficiently sensitive test for IHNV with a dynamic range that can help them make actionable decisions. Our initial product version will be further developed and improved upon through real-world trials as we prepare our system for commercialization.