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Applied and Environmental Microbiology, July 1999, p. 2942-2946, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

16S rRNA Gene Sequence Analysis of Photobacterium damselae and Nested PCR Method for Rapid Detection of the Causative Agent of Fish Pasteurellosis

Carlos R. Osorio,1,* Matthew D. Collins,2 Alicia E. Toranzo,1 Juan L. Barja,1 and Jesús L. Romalde1

Departamento de Microbiología y Parasitología and Instituto de Acuicultura, Universidad de Santiago de Compostela, 15706 Santiago de Compostela, Spain,1 and Department of Food Science and Technology, University of Reading, Reading RG6 6AP, United Kingdom2

Received 16 February 1999/Accepted 15 April 1999

The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.

* Corresponding author. Mailing address: Departamento de Microbiología y Parasitología, Facultad de Biología, Universidad de Santiago de Compostela, 15706 Santiago de Compostela, Spain. Phone: 34-981-563100, ext. 13255. Fax: 34-981-596904. E-mail: mpaetjlb{at}uscmail.usc.es.

Applied and Environmental Microbiology, July 1999, p. 2942-2946, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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