Anne Thebault

Detection of oyster herpesvirus DNA and proteins in asymptomatic Crassostrea gigas adults

Since 1972, several herpes-like virus infections have been reported among different bivalve species around the world. Most of these reports involved larvae or juveniles presenting high mortalities. Two case reports of herpes-like viruses concerned adult oysters, Crassostrea virginica in USA and Ostrea angasi in Australia. Molecular techniques including PCR and in situ hybridization (ISH) have been recently developed to detect the oyster herpesvirus genome. In the present study, 30 Pacific oyster, Crassostrea gigas, adults have been analyzed using three different techniques: PCR, ISH and immunochemistry, in order to detect herpesviruses in asymptomatic individuals. PCR and ISH allowed detection of oyster herpesvirus DNA in 93.3 and 86.6%, respectively, of analyzed oysters while polyclonal antibodies allowed detection of viral proteins in 76.6% of analyzed adult oysters. These results suggest that oyster herpesvirus infects adult oysters with high prevalence and that the virus may persist in its host after primary infection. The detection of viral DNA and viral proteins in the gonad of several individuals supports the hypothesis of a possible vertical transmission of the infection. Lastly, concordance among the three techniques used in this study is discussed.

Ostreid herpesvirus 1 detection and relationship with Crassostrea gigas spat mortality in France between 1998 and 2006

Since its molecular characterisation, Ostreid herpesvirus 1 (OsHV-1) has been regularly detected in Crassostrea gigas in France. Although its pathogenicity was demonstrated on larval stages, its involvement during mortality outbreaks at the juvenile stage was highly suspected but not evidenced. To investigate mortality outbreaks, the French National Network for Surveillance and Monitoring of Mollusc Health (REPAMO) carried out two surveys in juvenile C. gigas. The first survey lasted from 1998 to 2006 and was an epidemiological inquiry occurring when oyster farmers reported mortality outbreaks. The second survey, a longitudinal one, was set up in 1998 to complete the network observations on OsHV-1. Data analysis showed a specific pattern of mortality outbreaks associated with OsHV-1 detection. Ostreid herpesvirus 1 detection mainly appeared during the summer, suggesting the influence of the seawater temperature on its occurrence. It mostly presented a patchy distribution in the field in contrast to the nursery. Significant relationship between OsHV-1 detection and spat mortality was found, preferentially in sheltered and closed environments. The longitudinal survey confirmed most of the network observations. Although subsequent works particularly epidemiological surveys would be useful to confirm the causal link between the detection of OsHV-1 and the mortality outbreaks in juvenile C. gigas, the role of OsHV-1 in oyster mortality is progressing.

Whole clam culture as a quantitative diagnostic procedure of Perkinsus atlanticus (Apicomplexa, Perkinsea) in clams Ruditapes decussatus

Abstract The protozoan parasite Perkinsus atlanticus ( Azevedo, 1989 ) causes severe losses among cultured clams, Ruditapes decussatus . This parasite is routinely diagnosed by means of histology or incubation of gills in fluid thioglycollate medium. However, in order to develop models of experimental reproduction of the disease, a procedure for infection intensity evaluation was required. Thus, a diagnostic method has been developed, based on the culture of all clam tissues in fluid thioglycollate medium, followed by sodium hydroxide lysis, and iodine staining of the parasites on cellulose filters. This method was compared with histology. Results suggest that histology is not sensitive enough to detect low levels of infection. The whole-clam culture technique allows detection of low levels or early infection of clams by P. atlanticus . Moreover, this method provides a quantification of infection intensity as number of parasites per gramme wet weight tissue.

Relationship between pre- and post-metamorphic growth in the Pacific oyster Crassostrea gigas (Thunberg)

Twenty male and 20 female parental oysters, originating from four sites located along the French Atlantic coast, were crossed together. The 400 crosses were performed separately and then pooled to give a batch of larvae with a large genetic base. Successive sieving after 17 days at 23°C enabled the separation of the largest larvae from the batch. These larvae (i.e., sieving groups) were left to metamorphose and fix onto flat PVC collectors changed daily. Four groups of larvae representing distinctly different growth rates were successively separated during the attachment and metamorphosis of the whole population. This lasted 12 days, from the 17th day to the 29th after fertilisation. A part of each sieving group was settled separately as replicates on cultch. The post- metamorphic height was recorded weekly on 100 oysters per sieving group generating 400 growth curves. The oysters were removed from the collectors and weighed. The effect of the date of settlement (i.e., developmental rate) and sieving group (i.e., larval growth rate) affected (P<0.0001) the spat growth rate significantly. The correlation between sieving groups (i.e., larval growth) and spat growth rate was positive. After 11 months of growth under intensive conditions, the sieving group still had a significant effect on the total weight of juveniles settled on cultch (P<0.0001). These results justify the size-grading of larvae in bivalve hatcheries and show the great importance of early growth on growth in later stages in Crassostrea gigas.

Quantitative approach of risk management strategies for hepatitis a virus-contaminated oyster production areas.

It is not yet known whether using the new molecular tools to monitor hepatitis A virus (HAV) in shellfish production areas could be useful for improving food safety. HAV contamination can be acute in coastal areas, such as Brittany, France, where outbreaks of hepatitis A have already occurred and have been linked to the consumption of raw shellfish. A quantitative probabilistic approach was carried out to estimate the mean annual risk of hepatitis A in an adult population of raw oyster consumers. Two hypothetical scenarios of contamination were considered, the first for a rare and brief event and the second for regular and prolonged episodes of contamination. Fourteen monitoring and management strategies were simulated. Their effects were assessed by the relative risk reduction in mean annual risk. The duration of closure after abnormal detection in the shellfish area was also considered. Among the strategies tested, results show that monthly molecular reverse transcription PCR monitoring of HAV is more useful than bacterial surveys. In terms of management measures, early closure of the shellfish area without waiting for confirmatory analysis was shown to be the most efficient strategy. When contamination is very short-lived and homogeneous in the shellfish production area, waiting for three negative results before reopening the area for harvest is time wasting. When contamination is not well identified or if contamination is heterogeneous, it can be harmful not to wait for three negative results. In addition, any preventive measures, such as improving sewage treatment or producing shellfish in safer areas, that can reduce contamination by at least 2 log units are more efficient and less costly. Finally we show that controlling and managing transferred shellfish are useful and can play an important role in preventing cases. Qualitative results from HAV monitoring can advantageously supplement other measures that improve the safety of shellfish products in exposed areas.