The Phylogeny And Biological Characteristics Of Flavobacterium Columnare

Flavobacterium columnare, the agent for columnaris, is a world-wide bacterial pathogen, causing great economical losses in aquaculture. The infection of the bacterium may cause severe gill-rotting and sometimes skin ulceration. Bacterial gill-rot disease is epidemic in China, and it is also a major diseases of several leading cultured fish species in China, including the grass carp Ctenopharyngodoe idellus and the mandarin fish Siniperca chuatsi. The present research, by using bacteria isolated from outbreaks of columnaris disease in China as the pathogen, has studied the phylogenesis of Flavobacterium columnare, and the biological character of the bacterium, with the aim to provide an efficient way to prevent columnaris.To further understand the pathogenic mechanisam of Flavobacterium columnare, more bacterial pathogens were isolated from gill-rot diseased fish from fish farms in Hubei, Guangdong, Anhui, Sichuan provinces and in suburbs of Beijing city, and the five-step assessment of Flavobacterium columnare is applied well to these isolated strains. These characteristics, in combination with sequences of 16S rDNA, confirmed that the so-called gill-rot disease pathogen is actually the pathogen of columnaris disease, i.e. F. columnare. The bacteraium, Myxococcus piscicola Lu, Nie & Ko, 1975 named in 1970s as the pathogen of gill-rot disease of grass carp Ctenopharyngodon idellus is the synonym of F. columnare.Using the 16S rDNA sequences cloned from 16 strains of F. columnare in the present study, and some downloaded from the GenBank, a phylogenetic tree was established with the identification of three well-supported clades for these strains, corresponding clearly to reported three genomovars of this bacterium. The first clade contains only three strains, including the most virulent strain G4 isolated in 1970s from grass carp, and two others from Japan and USA, respectively. The second and third clades contain much more strains, including those from the same species of fish hosts, such as cyprinid fish. But strains isolated from salmonids are clustered in the second clade, with the inclusion of low-virulence strain G18 isolated in 1970s from grass carp in China. Strains isolated from the so-called Chinese perch, Siniperca chuatsi and Chinese sturgeon Acipenser sinensis in China are clustered in the third clade. This may, at least to some extent, indicate the difference in virulence of these strains; but their pathogenicity certainly requires further clarification.The biochemistry characteristics of the bacterium were also studied. The results show that Flavobacterium columnare has the ability to grow on the medium containing neomycin and tobramycin, to ferment saccharose, which suggests we could use neither neomycin nor SacB as screening marker while doing gene knock-out of the bacterium.Survival of Flavobacterium columnare after exposure to either pH 4.5, pH 9.2 or 0.8% NaCl, 1% NaCl, 2% NaCl for 15 min, 30 min or 1 h was studied in vitro. Just for 30 min, under any of the conditions the numbers of viable bacterial cells were significantly reduced. With pH 4.5, or the concentration of NaCl higher than 0.8% and the exposure time over 30 min, no grew on the medium. 24-32℃were the suitable temperatures for the bacterium. The fruiting body appeared when Flavobacterium columnare G₄ cultivated at 37℃, presumably it's a special state to adapt to new surroundings.By plate colony-counting methods, the relationship between optical density (OD₆₀₀) of the bacterial suspension and bacterial amount was evaluated. The optical density (x) and bacterial number(y) had a significant regression which could be expressed by the curve regression equation: y (G₄) =10⁹x^1.1653;y (Z4) =7×10⁸x^1.0356.