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The isolation, growth, and long-term storage of Flavobacterium branchiophilum is difficult. The aims of this work were to define optimal culture and storage media that would allow for growth of F. branchiophilum and determine if there were phenotypic and genotypic differences amongst isolates. Flavobacterium branchiophilum grew best on Anacker and Ordal’s medium that was supplemented with millimolar amounts of KCl, CaCl2, and MgCl2; with growth and colony visibility further improved with the addition of charcoal; 53 isolates were isolated from Ontario on this improved medium. Cultures of F. branchiophilum were viable over a period of 2 years when suspended in 5% (w/v) skim milk powder in Cytophaga Salts Broth or 10% DMSO in Cytophaga Salts Broth and stored at -75°C. By transmission electron microscopy, all isolates examined possessed surface structures resembling pili, and had evidence of membrane vesicles (MV) and tubules. The gyrB gene had a 7% difference in the 820 nucleotides sequenced, which was observed in 10 isolates from a particular case of BGD in brook trout and 4 other isolates. Differences between sequences of atpA and tuf from ONT case 6199 and 4 other isolates were also detected. Eventually, qPCR may be used to identify potential reservoirs of F. branchiophilum from the hatchery environment. |
