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...

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.

: Carp cathepsin L, which is the best candidate to produce textural change in the arai-treated carp Ðllet, exhibited maximum hydrolytic activity for Z-Phe-Arg-MCA and soluble casein at pH 5É0È5É5. The proteolytic action of the enzyme was evaluated by complete degradation of various carp myoÐbrils at pH 5É0 over 30 min and by potent degradation of the same proteins at pH 5É5È6É0 over 20 h. All myoÐbrillar components were partially degraded by the enzyme at pH 6É5È7É0, but varying amounts of them remained undegraded after 20 h. These Ðndings indicate that carp cathepsin L degrades not only carp myoÐbrillar components but also their resultant products between pH 5É0 and 7É0 and that it markedly acts on myosin heavy chain, a-actinin and troponin-T and -I. Carp cathepsin L likely contributes to postmortem muscle tenderisation of carp Ðllet over an extensive pH range during storage.1998 SCI. ( J Sci Food Agric 76, 499È504 (1998)

We document the potential of novel microsatellites as a genetic tool in furthering our understanding of the Crassostrea gigas genetic structure. From the microsatellite-enriched libraries we constructed, 123 repeat regions that had sufficient sequence information to design polymerase chain reaction primer sets were isolated. From these, 9 primer pairs were screened in a C. gigas population of 67 individuals to evaluate the genetic variability. All but 1 of the 9 loci showed allelic variation (number of alleles, 2-20; observed heterozygosity, 0.119-0.925; unbiased expected heterozygosity, 0.139-0.914). Considerable discrepancy of genotypic proportions from the Hardy-Weinberg equilibrium was observed at 1 locus with an apparent heterozygote deficiency. Several loci were successfully amplified in 3 other related species with the appropriate allele size: 6 loci in C. sikamea, 4 loci in C. ariakensis, and 5 loci in C. nippona.

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