WP3-T2 Study of pathogen survival outside the host through experimental trials

Participants :

  • 1 - IFREMER
  • 3 - UCC
  • 4 - CVI-IMARES
  • 7 - IRTA
  • 11 - ATLANTIUM

In order to complete information about the detection and the viability of pathogens from different sources in the environment, laboratory trials will be carried out.

The survival of selected pathogens in seawater and sediment will be studied over eitherhours, days or weeks through in vitro experiments using molecular, microbiological techniques and experimental infection trials (e. g. OsHV-1). Indeed, as no cell line is available at present to cultivate OsHV-1, the persistence of infection will be tested through experimental infection trials (Schikorski et al., in press).

The effects of variations in physico-chemical parameters (temperature, salinity, organic matter concentration, UV) will be also assessed using molecular, microbiological techniques, UV illumination bench scale (CBA) and experimental infection trials (e. g. OsHV-1). Concerning UV effects, laboratory based UV resistivity batch tests will be carried out in a first step by Participant 11 using bench scale technique based on Low Pressure And Medium pressure Collimated Beam Apparatus (CBA) on the relevant pathogens in order to define effective treatment conditions. Specific pathogens including Vibrio species and Nocardia crassostreae will be sent to Participant 11 and tested in order to define their specific UV resistivity and define the specific UV dose for the selected pathogens. Tests will be performed in Participant 11 laboratories.

In a second step, Participant 11 will manufacture and supply the UV devices (CBA devices) to Participants 1, 3, 4, and 7 and will prepare the protocols for UV illumination tests on the target pathogens for evaluating. Participant 11 will participate at part of the tests and will train the local researchers to operate the CBA devices. In addition Atlantium's researchers will participate in analysing the results and preparing the reports for generating the data for other WP's mainly WP5.

Vibrio growth rate and capability to enter the VBNC state in different matrices will be tested by evaluation of their culturability vs viability (Live Dead staining by epifluorescence microscopy) over time. Reactivation from the VBNC state inside bivalves will also be assayed. Presence of selected pathogenicity traits will be evaluated in culturable and VBNC cells. Since binding to surface allows bacteria to survive longer in sub-optimal
conditions, capability of selected Vibrio strains (reference strains and isolates from healthy and diseased molluscs) to interact with particles of chitin, which is the most abundant biopolymer in the marine environment, and zooplankton crustaceans (representative of the study area) will be tested. Adhesion efficiency will be analysed by the use of either radio-labelled or GFP labelled bacteria.

The presence in the adhesive strains of specific chitin binding ligands will be analysed by adherence inhibition experiments using substrate analogues (N-acetyl glucosamine and its oligomers). The presence of genes encoding for known ligands will be also verified. Culturability and viability of these bound bacteria will be also assayed. When this has been determined exposure trials will be carried out with naïve hosts to assess the pathogenicity of these agents after this length outside of the host.