WP2-T2 Search and identification of relevant pathogens

Participants :

  • 1 - IFREMER
  • 2 - CSIC
  • 3 - UCC
  • 4 - CVI-IMARES
  • 5 - IRTA
  • 6 - MI
  • 7 - UNIGE
  • 8 - UNIPD

Some characteristic sites (detection or absence of mortality events in previous years, presence of Pacific oysters and mussels) will be selected. Pacific cupped oyster and mussel samples will be collected and subjected to histology, molecular and culture-dependent analyses in order to detect the pathogens defined as relevant (OsHV-1, Vibrio splendidus, V. aestuarianus, Nocardia crassostreae and Marteilia refringens: pathogens associated to a disease and/or mortality outbreaks in Pacific oysters or mussels).

Mortality rates, selected environmental parameters (climate/weather, temperature, salinity, turbidity, oxygen, food availability, sea bed type, presence of blooms, lat/long/GPS identifier, type of production system) and animal parameters (species, age, sex, origin of stock, time since movement, population density, estimated mortality) vwill be also recorded. Information will be obtained about the presence/absence of the targeted
pathogens.

Mussel and Pacific cupped oyster sites known to be impacted or not by mortality outbreaks according to pre existing data from previous epidemiological surveys will be identified. Field sampling will include sites where disease outbreaks are known to occur (positive control) and where they are not known to occur. Sites will be selected (France, Ireland, Italy, Spain and The Netherlands) and monitored for increased mortality. Distinct ecosystems -lagoon versus open sea rearing areas- will be selected. Samples will be collected the first year and the second year of the project and tested for the presence of pathogens.

France :

3 sites will be selected along the French coast :

  • Thau lagoon,
  • Marennes Oléron (Atlantic coast)
  • Bay of Brest (Brittany)

Pacific oysters and mussels will be collected at the 3 sites and will be analysed to search OsHV-1, Vibrio species including V. splendidus and V. aestuarianus, Nocardia crassostreae and Marteilia refringens.

Spain :

  • 2 sites of the Spanish Mediterranean coast will be selected, being both bays of the Ebro River Delta : Alfacs Bay and Fangar Bays.
    • In Fangar Bay, Pacific cupped oysters will be collected to search for OsHV-1 and Vibrio species.
    • In Alfacs Bay, mussels would be the aquaculture targeted species in the search of Marteilia refringens; other shellfish species and various compartments (water column, sediment and zooplankton) will also be studied in Alfacs bay.
  • 2 sites will be also selected on the Spanish Atlantic coast where Crassostrea gigas and Mytilus galloprovincialis are reared.
    • The first sampling site will be the ría del Eo located in the Principado de Asturias (North of Spain). Crassotrea gigas was introduced in 1985, as spat to be grown on the intertidal area in cages. There are 3 administrative concessions for the culture. In this area there are also wild populations of Mytilus galloprovincialis that have not experienced mortalities and can be sampled.
    • The Ría de Vigo, the second site of sample collection, is located in the NW of Spain, situated at the south of Pontevedra province. The main cultured bivalve species is Mytilus galloprovincialis. No mortalities have been detected. The prevalence of Marteilia refringens is low. There also some Crasssostrea gigas that are kept temporarily either in depuration plants or in rafts befote sending them to the market.

The Netherlands :

2 sites will be selected where Shellfish cultivation takes place largely as bottom culture :

  • Oosterschelde has a restricted exchange of seawater with the North Sea
  • Lake Grevelingen is an artificially enclosed saltwater lake

Pacific oysters will be collected at the 2 sites and analysed to search for OsHV-1, Nocardia crassostreae and Vibrio species including V. splendidus and V. aestuarianus.

Ireland

3 sites will be selected from around the Irish Coast including sites which have and have not been impacted by the OsHV-1 μVar in 2009 and 2010 :

  • Donegal Bay,
  • Carlingford Lough,
  • Dungarvan Bay,

Pacific oysters will be collected to search for OsHV-1 and Vibrio species.

Italy :

  • The lagoon of Goro at the south of the Po River Delta (Adriatic Sea) is among the most important shellfish aquaculture systems in Italy and has been object of previous investigations. Exploiting natural and farmed populations, Mytilus galloprovincialis will be collected from a few selected sites of the area to investigate the presence of Vibrio species and other possible infective and parasitic agents. Crassostrea gigas has been reported inside the lagoon and is naturally present beyond Scanno di Goro (off-shore site).

Screening for selected pathogens will take place in different types of samples collected from France, Ireland, Italy, Spain and The Netherlands. Seasonality will play a strong role in sampling of OsHV-1 and Vibrio spp but sampling at least per season will be required for Marteilia refringens and Nocardia crassostreae. Thirty animals of the 3 species (if present at the selected sites) will be collected 6 times per year at least depending of the
culture practices, mortalities and pathogen presence. Monitoring of environmental parameters and sampling of animals will take place at least during the two first years of the project. Depending on the results, samples could also be collected in Year 3.

Bivalve samples (Pacific oysters and mussels) will be collected and analysed. Water, sediment, zooplankton, phytoplankton and macro fauna samples will be collected at the time of Pacific oyster and mussel collection. Other species potentially susceptible to the pathogens of interest can be collected. Mortality rates and selected environmental parameters (temperature, salinity, oxygen, chlorophylle A) will be also monitored.

To detect the various pathogens and possibly evaluate their physiological status, the samples will be subjected to culture-dependent assay, molecular analysis (real time-PCR, in situ hybridisation), histology (protozoans) and where appropriate, the ratio between culturable and total/viable bacteria will be evaluated; samples and/or isolates will be also screened for selected virulence-related traits (Vibrio, link with WP4) and culture of Nocardia crassostreae on specific plates will take place.

For Vibrio species, isolation, identification and counts from different compartments in the fields will be performed by culture and molecular methods, including APW enrichment, plating on TCBS, identification of isolates by standard methods and housekeeping gene sequencing. The presence of putative virulence traits will be tested by PCR. Analysis of the presence and concentration of total (culturable and non culturable) Vibrio species in the samples will be performed by real time PCR.

In order to test the hypothesis of sediment to be a scource for N. crassostreae, field experiments will be carried out to compare the prevalence of N. crassostreae in bottom cultured oysters compared to hanging methods of culture in an area with the presence of N. crassostreae.