Construction of a safe, stable, efficacious vaccine against Photobacterium damselae ssp. piscicida

Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine...

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Bibliographic Details
Published in:Diseases of aquatic organisms 2003-12, Vol.57 (1-2), p.51-58
Main Authors: THUNE, Ronald L, FERNANDEZ, Denise H, HAWKE, John P, MILLER, Ron
Format: Article
Language:English
Subjects:
3-Phosphoshikimate 1-Carboxyvinyltransferase
Alkyl and Aryl Transferases - genetics
Animal bacterial diseases
Animals
aroA gene
Bacterial diseases
Base Sequence
Bass
Biological and medical sciences
Electrophoresis, Agar Gel
Escherichia coli
Fish Diseases - immunology
Fish Diseases - prevention & control
Fish Diseases - virology
Gram-Negative Bacterial Infections - veterinary
Infectious diseases
Kanamycin Resistance - genetics
Medical sciences
Molecular Sequence Data
Morone chrysops
Morone saxatilis
Photobacterium - genetics
Photobacterium - immunology
Photobacterium damselae
Photobacterium damselae piscicida
Plasmids
Sequence Analysis, DNA
Vaccines
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Summary:Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This delta(aro)A::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.
ISSN:0177-5103
1616-1580
DOI:10.3354/dao057051