Flavobacterium columnare (formally Flexibacter columnaris) is a Gram negative, rod-shaped, aquatic bacterium which causes columnaris disease in warm and cold water species of fish. The bacterium is so named because wet mounts of F. columnare prepared from diseased fish appear as column-like, "haystack" colonies (Wakabayashi, 1991). The bacteria are detrimental pathogens, particularly in game fish raised in hatcheries. Diseases of fish, such as columnaris disease, are enhanced by stress factors such as unfavorable water temperature, crowding, injury, another disease, or improper fish husbandry. F. columnare causes columnaris disease at water temperatures between 18ƒ and 28ƒC but is rarely a problem at temperatures below 15ƒC. Columnaris disease is primarily a gill infection, but systemic infections are common (Amend, 1970). There are high and low-virulence strains of F. columnare (Rucker, et al., 1954). Low-virulence strains produce well advanced lesions on the gills or body surface or both, and often systemic infections occur. Several days may elapse before the fish dies from an infection caused by a low-virulence strain of F. columnare (Pacha and Ordal, 1967). High-virulence strains cause death within 24 to 48 hours after the fish contract the infection. In the case of high-virulence strains, lesions on the gills are small or not apparent, but some of the last fish to die have gray discoloration on the body surface most frequently around the dorsal fin in the form of an area that resembles a "saddle". Pacha and Ordal also observed that high-virulence strains infected fish and produced disease at lower water temperatures than low-virulence strains. In one experiment, high-virulence strains were able to produce mortalities at 12.8ƒC, whereas low-virulence strains were able to initiate infection only when the temperature was increased to 20ƒC (Pacha and Ordal, 1970).
Previous research has revealed that F. columnare possesses strong enzymatic activity and can degrade gelatin and chondroitin sulfates (Teska, 1993). Analysis of proteases produced by F. columnare has been performed by substrate gel electrophoresis with various substrates including gelatin and casein (Newton et al., 1997 and Bertolini and Rohovec, 1992). Proteases were detected based on their ability to digest the protein substrates incorporated into the acrylamide gels. The presence of a protease was indicated by a band of clearing in the blue background of the stained substrate (Newton et al., 1997). Another enzyme produced by F. columnare has been identified as chondroitin AC lyase (Griffin, 1991). Chondroitin AC lyase is an enzyme that degrades polysaccharides, particularly those found in cartilaginous connective tissue (Teska, 1993).
I propose to examine and compare the production of chondroitin AC lyase by several different strains of F. columnare obtained from the following sources: F. columnare cultures provided by the National Fish Research Laboratory in Kearneysville, WV, which were isolated from warm-water catfish; an American Type Culture Collection strain of F. columnare, #49512, which was originally isolated from a cold-water brown trout; and my own isolates that will be collected from various warm or cold-water New York State Fish Hatcheries; namely, the warm-water hatcheries at Oneida and Chautauqua, and the cold-water hatcheries at Rome, Salmon River, and Randolph. Presently, I have an isolate of F. columnare from the hatchery at Oneida, collected in October, 1998. The isolate was initially identified as F. columnare by the presence of "haystack" colonies evident on a wet-mount prepared from scrapings of a fathead minnow displaying grayish, "saddle" lesions around its dorsal fin. Scrapings from the grayish area were cultured on minimal nutrient agar plates. Colonies displaying yellow, rhizoid, spreading growth were isolated from the original mixed-culture plate and were confirmed to be F. columnare by a series of physiological and biochemical identification procedures, such as Gram stain; gelatin, casein, starch, and chondroitin A sulfate hydrolysis; oxidase, catalase, and hydrogen sulfide production; indole formation; and resistance to neomycin sulfate, polymyxin B sulfate, and tobramycin antibiotics.
My method of examining chondroitin AC lyase production will be a semi-quantitative analysis using the methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with chondroitin A sulfate incorporated into the acrylamide gel as the enzyme substrate. During SDS-PAGE, proteins (enzymes) isolated from a broth culture of F. columnare are loaded into an acrylamide gel that has been supplemented with the substrate, chondroitin A sulfate. After loading, an electric current supplied by two electrodes induces polarity along the gel length. The electric current through the gel causes the proteins to separate according to weight. Small proteins will migrate toward the bottom of the gel, while larger proteins will remain near the top of the gel. In this manner, proteins extracted from the bacterial cells can be identified according to their position in the gel after electrophoresis. The presence of chondroitin AC lyase in the gels will be indicated by appearance of a clear-zone of substrate degradation upon staining of the gel, which is expected to be located at 58-60 kDa (Bader, 1999). I hypothesize that the production of chondroitin AC lyase by the different strains of F. columnare will correlate with the origin and growth conditions of the isolates. F. columnare isolated from cold-water fish are expected to produce higher quantities of chondroitin AC lyase than F. columnare isolated from warm-water fish. Based on my comparisons of enzyme production by the various strains, I will inoculate bait minnows kept at warm and cold-water temperatures in an attempt to correlate the amount of enzyme produced with the subsequent virulence of each strain. Through further investigation of the differences in virulence of strains of F. columnare, scientists may be able to develop more effective methods for preventing columnaris disease in hatcheries where the bacterium causes high mortalities of fish.
REFERENCES
Bader, J. A. 1999. An inducible protease in Flavobacterium columnare: a possible role in virulence? Unpublished Data. The United States Department of Agriculture, Agricultural Research Service, Fish Diseases and Parasites Research Laboratory, Auburn, Alabama. 24th Annual Fish Health Workshop, 1999.
Bertolini, J. M., and J. S. Rohovec. 1992. Electrophoretic detection of proteases from different Flexibacter columnaris strains and assessment of their variability. Diseases of Aquatic Organisms. 12:121-128.
Griffin, B. R. 1991. Characteristics of a chondroitin AC lyase produced by Cytophaga columnaris. Transactions of the American Fisheries Society. 120:391-395.
Newton, J. C., T. M. Wood, and M. M. Hartley. 1997. Isolation and partial characterization of extracellular proteases produced by isolates of Flavobacterium columnare derived from channel catfish. Journal of Aquatic Animal Health. 9:75-85.
Pacha, R. E., and E. J. Ordal. 1967. Histopathology and experimental columnaris disease in young salmon. Journal of Comparative Pathology. 77:419-423.
Pacha, R. E., and E. J. Ordal. 1970. Myxobacterial diseases of salmonids. In: A Symposium on Diseases of Fishes and Shellfishes (ed. by S. F. Snieszko), pp. 243-257. American Fisheries Society, Special Publication No. 5, Washington, D. C.
Rucker, R. R., B. J. Earp, and E. J. Ordal. 1954. Infectious diseases of Pacific Salmon. Trans. Am. Fish Soc. 83(1953):297-312.
Teska, J. D. 1993. Assay to evaluate the reaction kinetics of chondroitin AC lyase produced by Cytophaga columnaris. Journal of Aquatic Animal Health. 5:259-264.
Wakabayashi, H. 1991. Effect of environmental conditions on the infectivity of Flexibacter columnaris to fish. Journal of Fish Diseases. 14:279-290.
Amend, D. F. 1970. Myxobacterial infections of salmonids: prevention and treatment. In: A Symposium on Diseases of Fishes and Shellfishes (ed. by S. F. Snieszko), pp. 258-265. American Fisheries Society, Special Publication No. 5, Washington, D. C.