No abstract

Flavobacterium psychrophilum is the agent of cold-water disease and rainbow trout fry syndrome in salmonid fish. Originally isolated in North Amenca only, the bacterium is now also responsible for severe mortalities in many salmonid hatcheries in Europe, as well as in Japan, Chile and Tasmania. The random amplified polymorphic DNA (RAPD) technique was used to analyze the genetic diversity among a collection of 177 F. psychrophilum strains isolated from different fish species and in different geographical areas. Forty 10-mer primers were tested and 5 of them were selected for further analysis of the bacterial DNAs. The primers OPH 06, OPH 08, OPG 08, OPG 14, and OPG 16 generated several reproducible profiles during a preliminary screening of the whole collection of stralns. Based on these results, the polyrnerase chain reaction (PCR) products of a selection of 60 bacterial DNAs were submitted to slow agarose gel electrophoresis for numerical analysis of the DNA fingerprintings. No correlation occurred between the combined RAPD profiles of the primers and the geographical origin of the strains, while some profiles were clearly associated with the fish species from which the strains were isolated. Another primer, OPG 10, yielded a unique RAPD profile common to all F. psychrophilum strains whereas the 9 other valld Flavobactenum species, several of which coexist in freshwater environments and may also be isolated from fish, displayed other profiles. Thus, depending on the primer used, both the typing of F. psychrophilum strains for epidemiology studies as well as the identification of this fish pathogen and its differentiation from related bacterial species could be achieved by using RAPD.

Aims:The cell envelope of the fish pathogen Flavobacterium psychrophilum contains more than 50 polypeptides resolved by sodium dodecyl sulphate-polyacrylaminde gel electrophoresis analysis including a major component named P60. Here, we have developed a simple and efficient procedure for the purification of P60 and therefore permitting its biochemical characterization. Methods and Results: Membrane proteins were selectively extracted from isolated cell envelopes with the mild non-ionic detergent Triton X-100. About 10 polypeptides were identified from the detergent fraction, including P60. The P60-enriched fraction was thereafter subjected to an anion exchange chromatographic step in the presence of Triton X-100. The molecule was purified at the milligram level (yield, about 75%; purification factor, 6.2). Analyses performed by charge shift electrophoresis, Triton X-114 phase separation and by detection of sugarmodified components showed that P60 is a true amphiphilic membrane-associated glycoprotein. Conclusions: The method described in this paper provides pure and non-denaturated P60 and should prove to be easily scaled-up. As sugar-modified protein, P60 should be included in the growing list of glycosylated prokaryotic proteins. Significance and Impact of the Study: It offers the possibility of obtaining P60 in amounts allowing the testing of the potential of P60 as a candidate for anti-flavobacteria subunit vaccines, as P60 is one of the major antigens.

The understanding of Fl. psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose.