The broad and vague phenotypic definition allowed the genus Pseudomonas to become a dumping ground for incompletely characterized polarly flagellated, Gram-negative, rod-shaped, aerobic bacteria, and a large number of species have been accommodated in the genus Pseudomonas. The 16S rRNA sequences of 128 valid and invalid Pseudomonas species, which included almost valid species of the genus Pseudomonas listed in the Approved Lists of Bacterial Names, were obtained : sequences of 59 species were determined and those of 69 species were obtained from the GenBank/EMBL/DD BJ databases. These sequences were compared with the sequences of other species of the Proteobacteria. Fifty-seven valid or invalid species including Pseudomonas aeruginosa (type species of the genus Pseudomonas Migula 1894) belonged to the genus Pseudomonas (sensu stricto). Seven subclusters were formed in the cluster of the genus Pseudomonas (sensu stricto), and the resulting clusters conformed well to the rRNA-DNA hybridization study by Palleroni (1984). The other species did not belong to the genus Pseudomonas (sensu stricto) and were related to other genera, which were placed in four subclasses of the

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)

On the basis of the 16S rRNA sequence data analysis among the closely related species, the specific primers for Cytophaga psychrophila were constructed.The specificity in amplifying the 16S rRNA of C.psychrophila was confirmed by using some selected strains of the related species. In addition, it was revealed that these specific primers distinguished C. psychrophila from other principal fish pathogens.The present PCR technique is expected to be a powerful tool for the diagnosis of cold-water disease.

A new Pseudomonas species, for which the name Pseudomonas plecoglossicida is proposed, was isolated from cultured ayu (Plecoglossus altivelis) with bacterial haemorrhagic ascites. The causative agent was similar to Pseudomonas putida biovar A in its phenotypic characteristics and on the basis of 16S rRNA gene sequence analysis, but it reduced nitrate to nitrite.

Genetic variability among 242 strains of Flavobacterium psychrophilum was characterized using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. Universal Primers GYR-1 and GYR-1R, which were designed to amplify the gyrase subunit B gene (gyrB), yielded a 1178 bp PCR product encoding gyrB and a 290 bp PCR product of anonymous DNA from all F. psychrophilum strains tested. In the RFLP analysis of the anonymous 290 bp DNA marker, the restriction enzyme Hin fI generated 2 cleavage patterns (Genotypes A and B). Genotype A was found only in isolates from ayu (n = 109), while Genotype B was found in isolates from coho salmon (n = 11), ayu (n = 35), rainbow trout (n = 43) and other fishes (n = 44). In the second experiment, Primers PSY-G1F and PSY-G1R specific for F. psychrophilum, were used to amplify gyrB. The specific primer pair amplified the expected size (1017 bp) PCR product from all F. psychrophilum strains. In the RFLP analysis of the gyrB, the restriction enzyme RsaI produced 2 genotypes, R and S. Genotype R was found in isolates from coho salmon (n = 6), ayu (n = 27), rainbow trout (n = 39) and other fishes (n = 4). Genotype S was found in isolates from coho salmon (n = 5), ayu (n = 117), rainbow trout (n = 4) and other fishes (n = 40). KEY WORDS: Genotyping · Flavobacterium psychrophilum · PCR-RFLP · Bacterial cold-water disease · gyrB gene Resale or republication not permitted without written consent of the publisherDis Aquat Org 56: [207][208][209][210][211][212][213][214] 2003 currently the most economically important fish disease in Japan.Some typing methods have been used to classify Flavobacterium psychrophilum strains for the epizootiological analysis of BCWD or RTFS. Serologic analysis , Lorenzen & Olesen 1997, Izumi & Wakabayashi 1999 and electrophoretic pattern of proteases (Bertolini et al. 1994), as well as genotyping using randomly amplified polymorphic DNA (RAPD) (Chakroun et al. 1997), ribotyping (Chakroun et al. 1998, Madsen & Dalsgaard 2000 and plasmid profiling , Chakroun et al. 1998, Madsen & Dalsgaard 2000 have been reported. In these genotyping studies of F. psychrophilum, however, the numbers of Japanese isolates tested were very small and do not seem to reflect the epizootiological situation of BCWD in Japan. The present study aimed to develop a new genotyping method for F. psychrophilum by restriction fragment length polymorphism based on PCR amplification (PCR-RFLP) of the gyrase subunit B gene (gyrB) and anonymous products, and its suitability for classifying 242 F. psychrophilum strains including 225 Japanese isolates from various host-fish species. MATERIALS AND METHODSBacterial strains and growth conditions. We examined 242 strains of Flavobacterium psychrophilum, comprising 11 strains from coho salmon, 144 strains from ayu, 43 strains from rainbow trout, and 44 strains from other species (amago Oncorhynchus rhodurus, yamame O. masou, iwana Salvelinus pluvins, grayling Thymallus thymallus, oikawa, common carp, ginbun...