Genotypic Diversity of Strains of Flavobacterium columnare from Diseased Fishes.

Abstract

Flavobacterium columnare, the causative agent of "columnaris disease" , is one of the most important bacterial pathogens in freshwater fishes (Wakabayashi, 1993). It was first isolated by Davis in 1922, and named Bacillus columnaris. The organism was variously reclassified as Chondrococcus columnaris (Ordal and Rucker, 1944), Cytophaga columnaris (Garnjobst, 1945) and Flexibacter columnaris (Leadbetter, 1974). The bacterium now belongs to Flavobacterium after Bernardet et al. (1996) amended the description of this genus. Song et al. (1988) found three types of colony morphology among Fl. columnare strains isolated from wide geographical areas, that is, rhizoid, mucoid and honeycomb-like type colonies. Anacker and Ordal for 18-20 h. 16S rDNA amplification The universal primers of 20F and 1500R were used to amplify 16S rDNA and their sequences are 5 '-AGAGTTTGATC(AC)TGGCTCAG-3' (position at 8 to 27 E. coli numbering) and 5'-CGATCCTACTTGCGTAG-3' (position at 1510 to 1492 E. coli numbering), respec tively (Weisburg et al., 1991). PCR amplification was performed in 50 ,ƒÊL containing 1.5 mM MgCl2, 0.5 nM of each dNTP (the nucleotide triphosphate of adenine, guanine, cytosine and thymine), 10 pM of each primer, 2.5 units of Taq polymerase and 400 ng of purified DNA. Samples were subjected to 30 cycles of amplification in a DNA thermal cycler (Gene AmpPCR System 2400, Perkin-Elmer). RFLP analysis PCR products were cut with restriction endonu cleases Haelll, Hhal, Mspl , Hinfl and Rsal. Digested fragments were separated by Mupid mini-gel electro phoresis aparatus with 3% NuSieve agarose. The results were detected by staining with 0.3-0.5% ethidium bromide for 30 min. 16S rDNA sequencing Sequencing of the PCR-amplified 16S rDNA was performed with the ABI Prism 377 DNA sequencer according to the protocol recommended by the manu facturer. Sequence data were compiled and binary sequence similarities were calculated with the GENETIX MAC (Software Development Co., Tokyo, Japan). The nucleotide substitution rate (Knuc values) (Kimura, 1980)

Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila

“…In contrast to Edwardsiella ictaluri, several studies have indicated distinctive genotypic diversity among F. columnare isolates (Triyanto & Wakabayashi 1999, Arias et al 2004. Despite the genetic polymorphism, primers designed by Welker et al (2005) were able to amplify DNA from all F. columnare fish isolates included in the study by Arias et al (2004) and the 10 F. columnare isolates when used in the m-PCR assay in this study.…”

Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge

“…It has a cosmopolitan distribution in temperate and tropical latitudes and has been isolated from an ecologically and phylogenetically diverse spectrum of wild-caught, aquaculture-reared, and ornamental freshwater fishes (Triyanto & Wakabayashi 1999, Schneck & Caslake 2006, Olivares-Fuster et al 2007. Given the nexus of appropriate host and environmental factors, columnaris disease can be economically devastating for the channel catfish Ictalurus punctatus (Rafinesque, 1818) aquaculture industry where in-pond mortality rates among adults and fingerlings can reach 60 and 90%, respectively (Plumb 1999).…”

Isolation and characterization of Flavobacterium columnare (Bernardet et al. 2002) from four tropical fish species in Brazil

“…The ubiquitous distribution of the organism in fresh water environments and the tendency for fish to acquire the disease after mechanical and/or environmental insults makes F. columnare among the most described pathogens in cultured, ornamental and wild fish populations (Triyanto and Wakabayashi, 1999;Austin and Austin, 1999;Bader et al, 2003;Jinu and Goodwin, 2004). Epizootics of columnaris are rarely spontaneous and have been troublesome in hatcheries, pen, cage culture and in pond culture, particularly under adverse environmental conditions and factors stressful to the fish.…”