Detection of Pathogenic Bacteria from Fishes and Their Environments by PCR

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(1) PCR primers specific for Cytophaga psychrophila, Cytophaga columnaris, Flexibacter maritimus, and Flavobacteium branchiophilum, were constraucted on the basis of the 16S rRNA sequence.(2) Carrier detection of C.psychrophila in ayu fry and coho salmon eggs was made by nested-PCR.In ayu sampled from the Biwa Lake, 35 percent (7/20) of the fish lots and 12 percent of the total fish examined (18/146) were positive in 1995, and 80 percent (4/6) of the fish lots and 33 percent (6/18) of the total fish were positive in 1996. In coho salmon eggs for aquaculture seedlings, five of seven lots from the wild spawner in USA were positive, and all four lots of eggs from domestic spawners in USA and Japan were negative.(3) For detection of Vibrio penaecida 16S rRNA-targeted RT-PCR was demonstrated to have 100 times higher sensitivity than 16S rDNA targeted PCR.Ten kuruma prawn for each sampling were collected at a private farm in Hiroshima Prefecter in 1996. Out of 100 prawn examined, ten prawn were positive in RT-PCR.(4) A 16S rRNA based RT-PCR method specific for Pasteurella piscicida was developed. The PCR detection of P.piscida in apparently healthy marine fishes in Ehime Prefecture had a high carrier state in both farmed and wild yellowtail.(5) Based on the nucleotide sequences of DNA fragments cloned from a plasmid pzP1 specific to P.piscicida, two primer sets were constructed. The PCR products were detected in DNA from the kidney of yellowtail infected naturally with P.piscicida, but not in that from healthy yellowtail.(6) A DNA fragment specific to Aerkomonas salmonicida subsp. salmonicida was cloned from the RAPD products. Based on the nucleotide sequence of the clone a PCR primer set was synthesized. It was effective in detecting A.salmonicida subsp. salmonicida from the kidney of experimentally infected amago salmon.