Submitted to: Journal of Aquatic Animal Health
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/23/2004
Publication Date: 8/17/2005
Citation: Shoemaker, C.A., Arias, C.R., Klesius, P.H., Welker, T.L. 2005. Technique for identifying Flavobacterium columnare using whole cell fatty acid profiles. Journal of Aquatic Animal Health 17: 267-274. Interpretive Summary: Columnaris disease, caused by the bacterium Flavobacterium columnare, is the second most important disease of the cultured catfish industry with diagnostic records suggesting more cases than enteric septicemia in recent years. Methods to identify F. columnare rely on biochemical tests and/or DNA methods (i.e., polymerase chain reaction (PCR)). Biochemical tests may require up to 14 days incubation time to determine the proper identification and PCR tests are costly. We have determined that the bacteria's fatty acid profile (substance in the bacteria's cell) is unique and can be used to positively identify F. columnare. A commercial system based on gas chromatography was used to generate the fatty acid profiles of F. columnare. The initial cost of the equipment is expensive, however, once in place in a diagnostic laboratory, extraction of the bacteria's fatty acids are inexpensive and results can be obtained in 24-48 h from bacterial isolates in pure culture. This method will provide a rapid and reliable method to identify F. columnare and other bacteria provided in the commercial database.
Technical Abstract: Isolates of Flavobacterium columnare (29 obtained from diseased fish and three ATCC cultures) were identified using biochemical characteristics prior to use in generating whole-cell fatty acid profiles. The microbial identification system (MIS; Microbial ID, Newark, DE), a gas chromatograpy system, was used to generate the fatty acid profiles of F. columnare. The MIS system contains databases of clinically and environmentally important pathogens that are represented by over 100 genera of bacteria including Flavobacterium (F. aquatile and F. mizutaii). Flavobacterium columnare is not in the MIS library, because it does not grow on standard media. Fatty acid profiles of F. columnare were generated using the CLIN40 protocol established by MIS following growth of the bacteria in modified Shieh broth. The fatty acid profiles of F. columnare isolates determined by the CLIN40 protocol included (greater than 1 percent): 13:0 ISO, unknown 13.57 (min), 15:1 ISO G, 15:0 ISO, 15:0 ANTEISO, 15:0, 16:0, 15:0 ISO 3OH, ISO 17:1 w9c, 18:1 w5c, 18:0, 17:0 ISO 3OH (92.6 % of profile). Five fatty acids were represented in the highest percentage from all isolates (CLIN40 method) and included: 15:1 ISO G (14.11%), 15:0 ISO (43.18%), 15:0 ISO 3OH (7.41 %), ISO 17:1 w9c (7.68 %) and 17:0 ISO 3OH (10.74 %). Analysis can be completed in 24 - 48 h provided the F. columnare isolates are in pure culture. Fatty acid profiles were also established using the MIS Rapid protocol (RCLIN50) in which identifications can be completed in 7 min instead of 20 min, the standard run time for the CLIN40 method. Fatty acids of the highest percentage for the RCLIN50 protocol included: 15:1 ISO G (11.38%), 15:0 ISO (28.64%), 16:0 (8.44%), 15:0 ISO 3OH (4.26 %), 18:1 w9c (4.46%), 18:1 w7c (3.16%), 18:0 (8.93%), 17:0 ISO 3OH (6.54%) and 10 methyl 16:0 (4.83 %). Results suggest that modified Shieh broth, Hsu-Shott's broth or modified Cytophaga broth can all be utilized to culture the bacteria for fatty acid analysis. This method will allow a rapid, inexpensive and reliable identification of F. columnare.