Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 29, 2002
Publication Date: March 20, 2003
Citation: BADER, J.A., SHOEMAKER, C.A., KLESIUS, P.H. RAPID DETECTION OF COLUMNARIS DISEASE IN CHANNEL CATFISH (ICTALURUS PUNCTATUS) WITH A NEW SPECIES-SPECIFIC 16-S R-RNA GENE-BASED PCR PRIMER FORFLAVOBACTERIUM COLUMNARE. JOURNAL OF MICROBIOLOGICAL METHODS.Vol.52,Issue 2,Pg.209-220, 2003. Interpretive Summary: Flavobacterium columare is the causative agent of columnaris disease. This disease is a cause of great economic losses in cultured catfish in the United States and in other cultured fish species worldwide. Annual losses attributed to columnaris disease are estimated at $50-60 million to the catfish industry. Presently, there are no vaccines to prevent columnaris disease in catfish, therefore disease is prevented through good management practices. In order to prevent disease through management practice, diseased fish must be detected early before mass infection occurs. This manuscript describes the development of such an early detection method, using a molecular-biology technique called the Polymerase Chain Reaction (PCR). The method described herein allows for rapid and early detection of the causative agent of columnaris disease, from agar plates, broth culture, frozen samples, dead fish tissue, and live fish in less than 5h or in 8h, if a preliminary product multiplication step (Nested PCR) is used. The PC product can be detected at low DNA concentrations and from as few as 100 bacterial cells. Nested PCR using a universal eubacterial primer can increase the sensitivity 5-fold down to 10 cells allowing detection of F. columnare strains in apparently healthy, asymptomatic fish. The efficiency of this diagnostic was compared to previously published PCR detection methods and was found to be better at early detection of columnaris disease under all conditions tested.
Technical Abstract: A 16 s rRNA gene from the chromosomal DNA of the fish-pathogenic bacteria Flavobacterium columnare (formerly Flexibacter columnaris), strain ARS-I, was cloned, sequenced and used to design a polymerase chain reaction (PCR) primer set. The primer set amplified a specific 1193 bp DNA fragment from F. columnare strains but not from related bacteria, F. pychrophilium, Flexibacter. maritimus, F. aquatile, F. branchiophilum, Cytophaga johnsonae; or unrelated bacteria, Escherichia coli, Edwardsiella ictaluri, E. tarda, Aeromonas hydrophila, and Streptococcus iniae. The PCR reaction conditions were optimized to permit detection of the organism from agar plates, broth culture, frozen samples, dead fish tissue, and live fish in less than 5 h (8 h, if nested PCR is used). DNA was extracted by a boiled- extraction method or by commercial column purification. The PCR product was detected at DNA concentrations below 0.5 ng and from as few as 100 bacterial cells. Nested PCR using universal eubacterial primers increased the sensitivity 5-fold, allowing detection of F. columare strains in apparently healthy, asymptomatic fish. The efficiency of this primer set was compared to the 16-S rRNA gene primer sets of Toyama et al. (1996) and that of Bader and Shotts (1998). The new primer set is as good or better than the previously published primer sets for detecting F. columnare in all samples and under all conditions tested.