Establishment of stable GFP-tagged Vibrio aestuarianus strains for the analysis of bacterial infection-dynamics in the Pacific oyster, Crassostrea gigas

Several marine pathogens are thought to be implicated in the summer mortality phenomenon that strikes the Pacific oyster stocks (Crassostrea gigas) in Europe since more than a decade. Although, since 2008, a herpes virus variant (microvar) is considered the main responsible for juvenile mortalities, the role of several associated bacteria is less clear. One of these, Vibrio aestuarianus, has often been detected in moribund oysters, and laboratory challenges proved its involvement in oyster death. However, the mechanisms by which this pathogen enters the oyster and transmits in-between specimens or collaborates with other pathogens remain thus far almost unknown. To establish genuine model strains, which allow the detection of the bacteria during the first hours of infection, both a highly pathogenic (02/41) and a weakly. pathogenic strain (01/308) were transformed with green fluorescent protein-expression vectors. The clones obtained were compared to the parental strains for their growth characteristics, basic metabolism, antibiotic-resistance and virulence. The 02/41 derivative was in all aspects indistinguishable from the parental strain. In contrast, in the 01/308 strain GFP expression led to a significant increase of virulence pointing to the dangers of GFP-tagging. The 02/41 GFP strain allows easy quantification by flow cytometry in both seawater and oyster haemolymph, and most importantly, its in situ detection will permit discerning the bacterium's routes inside the oyster tissues. (c) 2013 Elsevier B.V. All rights reserved.