Isolation and molecular characterization of Flavobacterium columnare strains from fish in Brazil

Abstract

Flavobacterium columnare, the etiologic agent of columnaris disease, has a broad geographical distribution and accounts for a large number of mortalities in fish species. This study aimed to generate a faster method for diagnosis of columnaris through isolation and characterization of the F. columnare 16S rDNA gene from bacteria isolated from Nile tilapia (Oreochromis niloticus), tambaqui (Colossoma macropomum) and matrinxa (Brycon amazonicus). The bacteria were characterized biochemically and by PCR-RFLP. For isolation, rasping with “swab” was performed directly on the characteristic lesions and the cephalic kidney of the fish then transferred to culture medium suitable for Flavobacterium. DNA was extracted for PCR and digestion with restriction enzymes. Altogether, 37 isolates were obtained. Biochemical assays included testing of absorption of Congo red, production of flexirrubin, production of H2S, nitrate reduction and motility. The results indicated that the isolates can be classified as F. columnare. The phylogram generated by the PCR-RFLP technique showed three main branches among of the F. columnare isolates. Therefore, the use of PCR-RFLP for identification of the bacteria was shown to be a more efficient and rapid tool than current biochemical techniques, which are time consuming and often inconclusive. Key words: Fish, Flavobacterium columnare, PCR-RFLP, 16S rDNA.