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Edwardsiella tarda is an important emergent pathogen in European aquaculture, causing several mortality events in turbot Scophthalmus maximus cultures in recent years. Here, we evaluated in parallel the specificity of 4 previously published pairs of primers, gyrBF1/gyrBR1, tardaF/ tardaR, etfA and etfD, for the detection of 53 E. tarda strains isolated from different sources, as well as 18 representatives of related and unrelated bacterial species. On the basis of the obtained results, we selected the pair of primers etfD, because it was the only one that recognized all E. tarda strains without false positive reactions. The sensitivity of this primer set showed detection limits of 2 cells per reaction tube in the case of pure cultures and 200 cells per reaction tube in mixed cultures. With regard to the sensitivity in seeded turbot tissues (kidney, liver and mucus), the detection limit was 3 x 10(2) E. tarda cells per reaction. In experimentally infected turbot, the etfD primer set was able to detect the pathogen in internal organs even 1 d post-infection, with a dose of 0.1 cells g(-1) of fish. In addition, this polymerase chain reaction protocol was useful for the detection of E. tarda in the field, and, based on the findings, we propose it as the most appropriate for accurate detection of E. tarda in routine diagnosis of edwardsiellosis in fish.

The 23S ribosomal RNA (rRNA) gene has been sequenced in strains of the fish pathogens Photobacterium damselae subsp. damselae (ATCC 33539) and subsp. piscicida (ATCC 29690), showing that 3 nucleotide positions are clearly different between subspecies. In addition, the 5S rRNA gene plus the intergenic spacer region between the 23S and 5S rRNA genes (ITS-2) were amplified, cloned and sequenced for the 2 reference strains as well as the field isolates RG91 (subsp. damselae) and DI21 (subsp. piscicida). A 100% similarity was found for the consensus 5S rRNA gene sequence in the 2 subspecies, although some microheterogeneity was detected as inter-cistronic variability within the same chromosome. Sequence analysis of the spacer region between the 23S and 5S rRNA genes revealed 2 conserved and 3 variable nucleotide sequence blocks, and 4 different modular organizations were found. The ITS-2 spacer region exhibited both inter-subspecies and intercistronic polymorphism, with a mosaic-like structure. The EMBL accession numbers for the 23S, 5S and ITS-2 sequences are: P. damselae subsp. piscicida 5S gene (AJ274379), P. damselae subsp. damselae 23S gene (Y18520), subsp. piscicida 23S gene (Y17901), P. damselae subsp. piscicida ITS-2 (AJ250695, AJ250696), P. damselae subsp. damselae ITS-2 (AJ250697, AJ250698).

The gene encoding the ferric uptake regulator protein (fur gene) of the fish pathogenic bacterium Photobacterium damselae ssp. piscicida Strain D121 was partially amplified using degenerate oligonucleotides. Complete sequencing of the fur gene and neighbouring DNA was accomplished by primer walking. An open reading frame of 447 bp, coding for a protein of 148 amino acids, and with high homology to previously described Fur proteins, was identified. The fur gene of P. damselae ssp. damselae ATCC 35083 was subsequently amplified by PCR with specific primers and its sequence determined, showing a 99.3% similarity to the P. damselae ssp. piscicida fur gene. The P. damselae fur gene was able to complement the fur mutation of Escherichia coli Strain H1681 in an iron-dependent fashion.

A PCR assay for detection and identification of the fish pathogen Listonella anguillarum was developed. Primers amplifying a 519 bp internal fragment of the L. anguillarum rpoN gene, which codes for the factor sigma54, were utilized. The detection limit of the PCR using L. anguillarum pure cultures was approximately 1 to 10 bacterial cells per reaction. For tissue or blood samples of infected turbot Scophthalmus maximus, the detection limit was 10 to 100 L. anguillarum cells per reaction, which corresponds to 2 x 10(3) to 2 x 10(4) cells g(-1) fish tissue. Our results suggest that this PCR protocol is a sensitive and specific molecular method for the detection of the fish pathogen L. anguillarum.

A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16S rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspecies piscicida and 15 isolates of subspecies damselae. With this procedure, all the P. damselae subsp. damselae strains yielded 2 amplification products, one of 267 bp and the other of 448 bp, corresponding to internal fragments of the 16S rRNA and ureC genes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16S rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNA probe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. damselae subsp. piscicida lacks this gene, whereas it is present in the subspecies damselae. This constitutes the first successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operon sequence, our results provided evidence that one of the steps in the P. damselae speciation proccess included gain/loss events associated with the ure operon.