Rapid detection of the fish-pathogenic bacterium pasteurella piscicida by polymerase chain reaction targetting nucleotide sequences of the species-specific plasmid pZP1

Abstract

A species-specific plasmid (pZP1) has been isolated from the fish-pathogenic bacterium Pasteurella piscicida. Two DNA fragments of PZP1 (PZP1-1 with 964 bp and PZP1-4 with 477 bp) were cloned from the plasmid pZP1. Two 20-mer primer sets, PZP1-1a and PZP1-1b, and PZP1-4a and PZP1-4b, were constructed according to the nucleotide sequences of fragments PZP1-1 and PZP1-4. A 484-bp DNA fragment was amplified from template DNA from 40 strains of P. piscicida by PCR using the PZP1-1a/1b primer set. The strains were isolated at different times of the year at different places in Japan and the USA. A 321-bp PCR product was amplified from all of the above strains of P. piscicida except strain ATCC17911 using the PZP1-4a/4b primer set. No such PCR products were obtained from template DNAs of Beneckea proteolytica, Photobacterium damsela, Ph. histaminum, Ph. leiognathi, 15 standard strains of Vibrio spp. or four fish pathogenic bacteria (Aeromonas hydrophila, A. salmonicida, Edwardsiella tarda and Enterococcus seriolicida). The PCR products were amplified with the PZP1-1a/1b primer set from DNA from the kidney of yellowtail (Seriola quinqueradiata) infected naturally with P. piscicida, but not from DNA from healthy yellowtail.