Characterization of abalone Haliotis tuberculata-Vibrio harveyi interactions in gill primary cultures
|Author(s)||Pichon Delphine1, Cudennec Benoit2, 3, Huchette Sylvain4, Djediat Chakib5, Renault Tristan3, Paillard Christine6, Auzoux-Bordenave Stephanie1, 7|
|Affiliation(s)||1 : Museum Natl Hist Nat, DMPA, UMR BOREA 7208 CNRS MNHN IRD UPMC, Stn Biol Marine, F-29900 Concarneau, France.
2 : Univ Lille 1, Lab ProBioGEM, F-59655 Villeneuve Dascq, France.
3 : IFREMER, Lab Genet & Pathol Mollusques Marins, Unite Sante Genet & Microbiol Mollusques, F-17390 La Tremblade, France.
4 : France Haliotis, F-29880 Plouguerneau, France.
5 : Museum Natl Hist Nat, F-75005 Paris, France.
6 : LEMAR Univ Bretagne Occidentale, CNRS UBO Ifremer IRD UMR6539, F-29280 Plouzane, France.
7 : Univ Paris 06, F-75005 Paris, France.
|Source||Cytotechnology (0920-9069) (Springer), 2013-10 , Vol. 65 , N. 5 , P. 759-772|
|WOS© Times Cited||12|
|Keyword(s)||Haliotis tuberculata, Vibrio harveyi, Gill cell culture, Pathogenicity, Phenoloxidase, Phagocytosis|
|Abstract||The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell—V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a nonpathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments.|