Flavobacterium columnare, a Gram-negative rod-shaped bacterium, is the causative agent of columnaris disease in a variety of fish hosts but is of particular significance to the catfish industry located in the southeastern United States. Columnaris infections are a leading cause of mortalities in catfish ponds, occurring alone or in conjunction with other diseases. Typical diagnostic methods for columnaris infections involve the use of selective media following the observation of gross signs of disease. A real-time quantitative PCR (qPCR) assay to estimate the quantity of bacteria present in environmental and tissue samples was developed and validated. The genetic variability seen in F. columnare makes detection of isolates from different genomovars (genetic groups) essential to an assay for diagnostic application. Isolates from catfish generally fall into one of two different genomovars, one being virulent to catfish, while the other genomovar is thought to be largely opportunistic. The qPCR assay described herein was designed specifically to detect F. columnare isolates from the two major genomovars most often associated with farm-raised catfish. The assay was shown specific to F. columnare, regardless of genomovar, and demonstrated sensitivity consistent with similar qPCR assays. In addition, the assay provides quantitative information, estimating the bacterial loads in fish tissue and the environment. Two different applications of the assay are presented: (1) Estimate bacterial burden in fish tissue following immersion challenges to identify variation in transmission rates between channel and blue x channel hybrid catfish, and (2) Estimate the environmental burden of F. columnare in catfish ponds over the course of a single calendar year. This assay will provide an invaluable tool for researchers and diagnosticians in expanding our understanding of F. columnare and how it interacts with the host and environment
