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

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.