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Applied and Environmental Microbiology, May 2004, p. 2702-2708, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2702-2708.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Behavior of an Aeromonas hydrophila aroA Live Vaccine in Water Microcosms

José Vivas,¹ Begoña Carracedo,¹ Jorge Riaño,¹ Blanca E. Razquin,¹ Pilar López-Fierro,¹ Félix Acosta,² Germán Naharro,³ and Alberto J. Villena^1*

Department of Cell Biology, Faculty of Biological and Environmental Sciences,¹ Department of Animal Pathology (Animal Health), Veterinary Faculty, University of León, 24071 León,³ Department of Animal Pathology, Veterinary Faculty, University of Las Palmas de Gran Canaria, Arucas, Spain²

Received 11 December 2003/ Accepted 20 January 2004

Genetically modified auxotrophic mutants of different fish pathogens have been used as live vaccines in laboratory experiments, but the behavior of the strains after release into aquatic ecosystems has not been characterized. We previously constructed and characterized an aroA mutant of Aeromonas hydrophila and studied the protection afforded by this mutant as a live vaccine in rainbow trout. In this work, we describe the survival of this strain in aquatic microcosms prepared from fish water tanks. The aroA mutant disappeared rapidly in nonfiltered, nonautoclaved fish tank water, declining below detection levels after 15 days, suggesting an inhibitory effect of the autochthonous microflora of the water. When the aroA strain was used to inoculate sterilized water, its culturability was lower than that of wild-type strain A. hydrophila AG2; after long periods of incubation, aroA cells were able to enter a viable but nonculturable state. Entry into this nonculturable state was accompanied by changes in the cell morphology from rods to spheres, but the cells appeared to remain potentially viable, as assessed by the preservation of cell membrane integrity. Supplementation of the culture medium with sodium pyruvate favored the culturability and resuscitation of the two A. hydrophila strains at low temperatures (6 and 16°C). These results contribute to a better understanding of the behavior of the aroA strain in natural environments and suggest that the inactivation of the aroA gene may be beneficial for the safety of this live vaccine for aquacultures.

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* Corresponding author. Mailing address: Department of Cell Biology, Faculty of Biological and Environmental Sciences, University of León, 24071 León, Spain. Phone: 34 987 291 487. Fax: 34 987 291 487. E-mail: dbcavc{at}unileon.es.

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Applied and Environmental Microbiology, May 2004, p. 2702-2708, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2702-2708.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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