Experimental ostreid herpesvirus 1 infection of the Pacific oyster Crassostrea gigas: kinetics of virus DNA detection by q-PCR in seawater and in oyster samples.

Herpes- and herpes-like viruses are known to infect a wide range of bivalve mollusc species throughout the world. Abnormal summer mortalities associated to the detection of ostreid herpesvirus 1 (OsHV-1) have been currently reported in France among larvae and spat of the Pacific cupped oyster Crassostrea gigas. In the present work, we have developed an experimental protocol of horizontal transmission based on the cohabitation between healthy and experimentally infected oysters. Through a cohabitation trial, the kinetics of OsHV-1 detection in different oyster organs and seawater samples were investigated and characterized for the first time using real time quantitative PCR.

Rapid and sensitive detection of ostreid herpesvirus 1 in oyster samples by real-time PCR.

Herpes and herpes-like virus infections have been reported in various marine mollusc species associated with high mortality rates. Following the characterisation and genome sequencing of ostreid herpesvirus 1 (OsHV-1), specific diagnostic tools have been developed based on conventional PCR techniques or in situ hybridisation. We have now developed a real-time PCR assay for rapid, sensitive and quantitative detection of OsHV-1, and compared it with a conventional PCR technique described previously. The new assay utilised SYBR((R)) Green chemistry with specific primers C(9)/C(10) targeting the C region. The melt curve analysis of OsHV-1 DNA or DNA extracted from infected material showed only one melting temperature peak (75.75+/-0.1 degrees C). The assay had a detection limit of 4 copies/microL of viral genomic DNA and a dynamic range of 5 logs. Using infected oyster samples as template, the assay was about 100-fold more sensitive than single PCR method using C(2)/C(6) primers. The assay was applied successfully for rapid diagnosis (100 min) and quantitation of OsHV-1 in different developmental stages of Crassostrea gigas. Although it already exists a competitive PCR method to quantify OsHV-1 DNA, quantitative data that will emerge in future using the new sensitive and reliable assay will illuminate aspects of pathogenesis, in particular the viral loads in asymptomatic oysters and the kinetics of infection in specific target tissues.

Husbandry stress during early life stages affects the stress response and health status of juvenile sea bass, Dicentrarchus labrax.

In aquaculture management it is important to establish objective criteria to assess health and welfare of the fish. Here we show that European sea bass (Dicentrarchus labrax) confronted with husbandry-associated stress (tank cleaning, i.e. scrubbing, and water temperature variation) during early life stages show poorer survival and disease resistance as juveniles. We evaluated several parameters for stress (plasma cortisol, glucose and lactate, hydromineral status), growth performance, the immune response (plasma IgM levels) and the effects of a nodavirus challenge. Principal component analysis allowed the establishment of a stress panel including plasma cortisol, osmolality, IgM levels and weight. Sea bass juveniles reared during early life in high and constant temperature perform best in terms of stress-related parameters assessed by principle component analysis. Variable water temperature triggers dramatic changes in plasma cortisol, osmolality, IgM levels, body weight and susceptibility to nodavirus that suggest a strong and prolonged activation of the HPI axis. Scrubbing induces some disturbances typical for mild short-term, acute stress, viz. increased plasma osmolality and decreased IgM levels, but does not affect plasma cortisol, growth or susceptibility to nodavirus of sea bass. Our data fit well with the concept of allostasis. We discuss the relevance of our work for sea bass aquaculture.

Sea bass Dicentrarchus labrax nervous necrosis virus isolates with distinct pathogenicity to sea bass larvae.

Reproduction of nodavirus disease was performed by experimental infection of sea bass eggs during fertilization or at larval stage 4 with 2 genetically distinguishable nodavirus strains (Sb1 and Sb2) isolated from sea bass collected along the Atlantic and Mediterranean French coast. The pathogenicity of the virus strains was assigned after detection of the virus by ELISA and immunohistochemistry (IHC). The Atlantic (Sb1) strain was more pathogenic than the Mediterranean (Sb2) strain during the fertilization step whilst both strains were pathogenic following experimental exposure of 4 d old larvae. Virus lesions developed in the brain 4 to 6 d following experimental exposure. Experimental ELISA proved very sensitive for detecting the nodavirus in Sb1 or Sb2 experimentally infected larvae, as well as in naturally infected sea bass larvae collected in French hatcheries or in barramundi larvae reared in the Pacific area. The development of an ELISA specific for the 2 nodavirus strains isolated from the sea bass should be useful for the detection of the virus, in addition to other techniques recommended by the Office International des Epizooties (OIE).