Cytotechnology

, Volume 65, Issue 5, pp 759–772 | Cite as

Characterization of abalone Haliotis tuberculataVibrio harveyi interactions in gill primary cultures

  • Delphine Pichon
  • Benoit Cudennec
  • Sylvain Huchette
  • Chakib Djediat
  • Tristan Renault
  • Christine Paillard
  • Stéphanie Auzoux-Bordenave
Marine Invertebrate Cell Culture

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 non-pathogenic 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.

Keywords

Haliotistuberculata Vibrioharveyi Gill cell culture Pathogenicity Phenoloxidase Phagocytosis 

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Delphine Pichon
    • 1
  • Benoit Cudennec
    • 2
    • 5
  • Sylvain Huchette
    • 3
  • Chakib Djediat
    • 4
  • Tristan Renault
    • 5
  • Christine Paillard
    • 6
  • Stéphanie Auzoux-Bordenave
    • 1
    • 7
  1. 1.Station de Biologie MarineMuséum National d’Histoire Naturelle, DMPA, UMR BOREA 7208 CNRS/MNHN/IRD/UPMCConcarneauFrance
  2. 2.Laboratoire ProBioGEMUniversité Lille 1Villeneuve-d’AscqFrance
  3. 3.France-HaliotisPlouguerneauFrance
  4. 4.Plateforme d’Imagerie et de Microscopie Electronique (PIME)Muséum National d’Histoire NaturelleParisFrance
  5. 5.Unité Santé, Génétique et Microbiologie des Mollusques—Laboratoire de Génétique et Pathologie des Mollusques MarinsIfremerLa TrembladeFrance
  6. 6.UMR6539 CNRS/UBO/Ifremer/IRD, LEMAR Université de Bretagne OccidentalePlouzanéFrance
  7. 7.Université Pierre et Marie Curie Paris VIParisFrance

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