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...

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Flavobacterium psychrophilum is the causative agent of the fish diseases called bacterial cold-water disease and rainbow trout fry syndrome. It has been reported that some isolates of F. psychrophilum are resistant to quinolones; however, the mechanism of this quinolone resistance has been unexplained. In this study, we examined the quinolone susceptibility patterns of 27 F. psychrophilum strains isolated in Japan and the United States. Out of 27 isolates, 14 were resistant to both nalidixic acid (NA) and oxolinic acid (OXA), and the others were susceptible to NA and OXA. When amino acid sequences deduced from gyrA nucleotide sequences of all isolates tested were analyzed, two amino acid substitutions (a threonine residue replaced by an alanine or isoleucine residue in position 83 of GyrA [Escherichia coli numbering] and an aspartic acid residue replaced by a tyrosine residue in position 87) were observed in the 14 quinolone-resistant isolates. These results strongly suggest that, as in other gram-negative bacteria, DNA gyrase is an important target for quinolones in F. psychrophilum.

The nested PCR was used to detect Cytophaga psychrophila carried by juvenile ayu (Plecoglossus altivelis) and eyed eggs of coho salmon (Oncorhynchus kistch). Ayu juveniles were caught in Lake Biwa and frozen shortly after landed. The coho salmon eggs from the USA and Japan were collected on a lot-by-lot basis at Narita airport or hatcheries when the containers arrived. Chelex 100 was used as a medium for extraction of DNA from these samples. Utilizing the PCR, a specific primer pair of PSY1 and PSY2 for C. psychrophila was used to amplify its 16S rDNA segments. An amplification product of the expected size was obtained from 5 of 24 DNA extractions from the kidney of ayu samples in April 1995. These fish had been previously diagnosed as negative for the presence of C. psychrophila by cultivation on TYE agar and IFAT. Five of 11 lots of coho salmon eggs were found to be positive for C. psychrophila by PCR. Four of these five lots had also been diagnosed as positive by cultivation and IFAT. In addition, the sensitivity of the PCR amplification for detecting C. psychrophila in kidney samples of ayu was calculated to be 1.5 CFU per PCR reaction tube.

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