At La Mortanne from 2 to 7 June, viral-DNA amounts of ≥10 4 DNA copies per mg wet tissue (category 2) were detected in some oysters ( Fig.
3a and b and Table 1) without a concomitant increase in the mortality rates. An increase in the percentage of positive animals was reported on 26 April at La Mortanne and Les Sables de l'Are ( Fig. RESULTS Water temperature and oyster mortality.Īt both field sites (La Mortanne and Les Sables de l'Are), viral-DNA amounts remained below 10 4 DNA copies per mg of wet tissue until 31 March to 26 May, a period during which the percentage of OsHV-1-positive individuals was less than 100% ( Fig. The results were expressed as the viral-DNA copy number mg wet tissue −1. Melting curves were plotted (55 to 95☌) in order to ensure that a single specific PCR product was amplified. The specificity of the PCR products was systematically confirmed based on the melting-temperature value calculated from the dissociation curve. Briefly, the protocol used SYBR green chemistry with specific primers, DPFor/DPRev, targeting the region of the OsHV-1 genome predicted to encode a DNA polymerase catalytic subunit ( 31). The detection and quantification of OsHV-1 DNA was carried out using a previously published real-time PCR protocol ( 30). More than 1,000 real-time PCR analyses were carried out.
Oyster spat mortality rate trial#
It was chosen to collect samples with very great frequency during the trial (around 30 dates of collection) and to conduct OsHV-1 DNA detection individually on 10 oysters for each sample date and site. The laboratory takes part in a yearly proficiency test (interlaboratory comparison) organized by the national reference laboratory for marine mollusc diseases (Ifremer, La Tremblade, France). A causal link between OsHV-1 infection and oyster spat mortality has been suggested in different epidemiological studies ( 27 – 29).Īnalyses were carried out by Genindexe (La Rochelle, France), a laboratory officially recognized by national authorities (the French Ministry of Agriculture) for OsHV-1 detection in C. However, the demonstration of a causal link between a pathogen and mortality outbreaks in the field may not be exclusively based on the possibility of reproducing the disease experimentally but should take into account the idea that the cause of mortality outbreaks may be a combination of factors. In addition to transmitting OsHV-1 infection by intramuscular inoculation, waterborne transmission to healthy oyster spat can also occur via cohabitation of healthy oyster spat with experimentally infected individuals ( 26). In this context, OsHV-1 was inferred to be the causative agent of the mortality reported in the study ( 25). Furthermore, analysis of injected oyster spat revealed large amounts of OsHV-1 DNA by real-time quantitative PCR. The results of experimental trials showed that mortality was induced after the injection. During this period, the water temperature was below 16☌.Ī protocol based on the intramuscular injection of 0.22-μm-filtered tissue homogenates prepared from naturally infected spat was developed ( 25). An initial increment in the number of PCR-positive oysters was reported at both field sites during the survey in the absence of significant mortality. Mass mortality was observed among oysters a few days after increases in the number of PCR-positive oysters and viral-DNA amounts were recorded. This difference may be related to earlier increases in water temperature. Although similar massive mortality rates were reported at the 4 sites, mortality was detected earlier in the pond and in the nursery than at both field sites. Mortality rates were recorded based on regular observation, and samples were collected to search for and quantify OsHV-1 DNA by real-time PCR. For this purpose, a single batch of Pacific oyster spat was deployed in 4 different locations in the Marennes-Oleron area (France): an oyster pond (“claire”), a shellfish nursery, and two locations in the field. Additional data are necessary to better describe mortality events in relation to environmental-parameter fluctuations and OsHV-1 detection. gigas in Europe have been related to the detection of a variant of OsHV-1 called μVar. Since 2008, mass mortality outbreaks among C. This virus has been classified as Ostreid herpesvirus 1 (OsHV-1) within the family Malacoherpesviridae. A particular herpesvirus was purified from naturally infected larval Pacific oysters, and its genome was completely sequenced. A number of bivalve species worldwide, including the Pacific oyster, Crassostrea gigas, have been affected by mass mortality events associated with herpesviruses, resulting in significant losses.