Development of a High Sensitivity and Specificity Quantitative Aptamer Assay for Coldwater Disease Management Applications

NON-TECHNICAL SUMMARY: High morbidity and mortality from infection with F. psychrophilum, the causative agent of Coldwater Disease and rainbow trout (Oncorhynchus mykiss) fry syndrome, have become significant problems for commercial and conservation aquaculture operations worldwide. First described in 1948 in the United States, the disease caused by this bacterium has been identified throughout North America, Europe and other countries. There is strong evidence that F. psychrophilum is transmitted vertically; thus, it has been hypothesized that disease management at hatchery facilities can be improved through broodstock screening and implementation of culling programs. A low-cost highly sensitive and specific diagnostic assay for Coldwater Disease (Flavobacterium psychrophilum infection) is proposed. This assay will have applications for water samples or fish tissue. Infoscitex will develop high affinity aptamers (synthetic DNA oligonucleotides) capable extremely high sensitivity detection of the pathogen (bacteria) or material deriving from the pathogen. The developed aptamer will be used in place of antibodies to detect with greater sensitivity the pathogen and quantify its concentration by quantitative real-time polymerase chain reaction (qRT-PCR). Alternative ELISA based assays utilizing such aptamers will also be developed. A higher specificity and selectivity aptamer based assay will provide US fish farmers and fish health laboratories with the ability to perform earlier detection of the onset of CWD or allow quantitative screening of broodstock in order to implement strategies that would limit vertical transmission to progeny. This will enable early (ideally pre-epidemic) treatment of fish or culling of eggs from infected broodfish and prevent or limit inevitable losses by the US aquaculture industry. The ability to cull or remove infected fry will result in significant savings for the industry. F. psychrophilum can result in considerable economic losses due to high mortality in fry and juvenile fish. However, economic losses can also stem from product quality problems from skin ulcerations and vertebral deformation in larger fish, leading to the degradation of the sale price as they go to market. In addition, significant cost savings will come from avoiding the labor cost associated with raising infected fish. Another subtle but important economic impact is the increased susceptibility to other pathogens and the need for costly chemotherapeutics. Some estimates put the economic cost of this type of coldwater disease at tens of millions annually, among commercial aquaculture producers and conservation hatcheries. If our aptamer based detection assay is expanded to include other cold water diseases such as Flavobacterium columnare, which impacts catfish and other cultured fish, total worldwide economic cost savings may exceed $100 million annually. <P>APPROACH: Infoscitex has developed a novel proprietary one step technique for aptamer production which yields aptamers with extremely high affinity and specificity for the target. From two Phase II SBIR programs funded by the DOD, affinities of 10-18 M (Kd) have been obtained using aptamers produced by to detect infectious prions (Chronic Waste Disease) and anthrax toxin. This sensitivity allows for the dramatic lowering of the detection limits for various biomolecules, and as a result, a very early diagnosis of infections. The overall objective of this Small Business Innovation Research (SBIR) program is to develop an aptamer-based assay for the early detection of F. psychrophilum in a common sensor platform, such as ELISA or real-time PCR. In Phase I, Infoscitex and the University of Idaho will use the real-time PCR and ELISA laboratory assays to confirm the feasibility of the selected approach. The product of a successful Phase I program will be the development of aptamer with high selectivity and specificity for F. Psychrophilum. The sensitivity and selectivity levels of this aptamer will be characterized to demonstrate its advantages over current antibody tests. In addition, a successful Phase I will yield an aptamer-based assay which should detect F. Psychrophilum at much lower levels than previously achievable. This ability will allow for studies to be conducted to understand the effects on fish and fry of various levels of F. Psychrophilum concentrations in water and fish tissue. Infoscitex, in collaboration with Dr. Cain at U-Idaho, will conduct evaluations to determine the relationship of bacteria concentration in water and tissue to the onset of disease in individual fish as well as to the time of propagation to the population. The data from this type of study will allow researchers and fish farmers to determine the severity of their infection and what level treatment should be implemented. In Phase II, the assays will be integrated for use on traditional, well-established, qRT-PCR and ELISA platforms. This effort will be conducted in consultation with a commercial entity in the aquaculture industry to ensure that the design of the assay matches current aquaculture practice. On such company, Western Chemical has been contacted to discuss their possible involvement in this program and they have expressed interest in the potential to take the product of this work to commercialization. At the conclusion of Phase II, we anticipate that the aptamer assay will be fully characterized and ready for license to our commercialization partner, which will limit the cost of research and development for this assay to under $500,000.