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
Introduction
Infections by herpes- or herpes-like viruses have been reported in various marine mollusc species throughout the world. Such viruses were reported in USA (Farley et al., 1972, Friedman et al., 2005), New Zealand (Hine, 1992), Australia (Hine and Thorne, 1997), Mexico (Vasquez-Yeomans et al., 2004), in Asian countries (Moss et al., 2007) and France. The first description of a virus morphologically similar to members of the Herpesviridae family was reported by Farley et al. (1972) in eastern oysters Crassostrea virginica. Herpes-like virus infections have then been reported in other bivalve species, including the Pacific oyster Crassostrea gigas (Arzul et al., 2001b, Arzul et al., 2001c, Hine, 1992, Nicolas et al., 1992, Renault et al., 1994), the European flat oyster Ostrea edulis (Arzul et al., 2001b, Arzul et al., 2001c, Comps and Cochennec, 1993, Renault et al., 2000b), the Chilean oyster Tiostrea chilentis (Hine et al., 1998), the Portuguese oyster Crassostrea angulata (Arzul et al., 2001b), the Suminoe oyster Crassostrea rivularis (Arzul et al., 2001b, Arzul et al., 2001c), the Manila clam Ruditapes philippinarum (Arzul et al., 2001c, Renault and Arzul, 2001, Renault et al., 2001), the carpet shell clam R. decussatus (Arzul et al., 2001c, Renault and Arzul, 2001), and the French scallop Pecten maximus (Arzul et al., 2001a). Herpes-like infections have also been described recently in different abalone species Haliotis laevigata, Haliotis rubra, and Haliotis diversicolor (Chang et al., 2005, Hooper et al., 2007, Tan et al., 2008).
Since 1991, episodic herpes-like virus infections have been reported in France in Crassotrea gigas spat and juveniles (Renault, 1998). These virus infections are mostly evidenced during the summer period, concomitantly with a rapid increase of seawater temperature (Garcia et al., unpublished data). They are generally associated with high mortality rates reaching up to 80% (Renault et al., 1994, Renault et al., 1995). Herpes-like viruses have also been detected in adult oysters (Arzul et al., 2002, Hine and Thorne, 1997). However, adults appear less sensitive to the disease than spat. This may be explained in part by the existence of a higher antiviral immune response in adults. Previous studies have presumed the presence of hypothetical antiviral molecules in Pacific oyster haemolymph (Olicard et al., 2005a, Olicard et al., 2005b).
A purification protocol established in order to isolate viral particles from infected C. gigas larvae allowed the characterization of the complete genome sequence and the nucleocapsid reconstructed structure of the virus (Davison et al., 2005, Le Deuff and Renault, 1999). This virus was classified as the sole member of the genus Ostreavirus (family Malacoherpesviridae, order Herpesvirales) and was named ostreid herpesvirus 1 (OsHV-1) (Arzul et al., 2002, Davison, 2002, Davison et al., 2005, Davison et al., 2009, Minson et al., 2000).
The economical importance of C. gigas oyster aquaculture has contributed to the development of specific and sensitive diagnostic tools, including polymerase chain reaction (PCR) (Renault et al., 2000a, Renault and Lipart, 1998), in situ hybridization (ISH) (Lipart and Renault, 2002, Renault and Lipart, 1998), immunohistochemistry (Le Deuff et al., 1995), and recently real-time PCR (Pepin et al., 2008). These techniques allowed to keep a watch on the detection of the viral infection in the natural environment through epidemiological investigations. Furthermore, the infectivity of OsHV-1 was confirmed towards younger stages of C. gigas through experimental trials conducted in laboratory conditions. It was first demonstrated that axenic healthy larvae could be infected by contact with filtered suspensions of ground infected larvae (Arzul et al., 2001b, Arzul et al., 2001c, Le Deuff et al., 1995, Le Deuff et al., 1996).
In 2008, in order to explore the causes of massive mortality outbreaks affecting French C. gigas spat, experimental infection trials were carried out (Schikorski et al., submitted for publication). The intramuscular injection of viral suspensions prepared from naturally infected fresh oysters induced high mortality rates reaching up to 80% in 3 days in healthy 1-year-old oysters. The capacity to obtain OsHV-1 experimentally infected oysters allowed us to develop a protocol of horizontal transmission through cohabitation trials. It was demonstrated that healthy oysters after 2 days of cohabitation with experimentally infected oysters develop the disease. Thus, our previous results contributed to the set up of a reproducible model for studying the pathogenicity of OsHV-1 on C. gigas oysters in experimental conditions.
In this context, the aim of the present study was to investigate the kinetic of OsHV-1 DNA detection in the Pacific oyster C. gigas in cohabitation assays between healthy and experimentally infected oysters. The experiment was conducted in a closed system without the seawater being changed. Virus DNA quantification was performed by real-time quantitative PCR (qPCR) in the time course of the experiment in different oyster tissues including haemocytes, gills, mantle, adductor muscle, and digestive gland. Besides, very little information is available concerning the detection of the virus in seawater. Thus, virus DNA amounts were also measured in seawater during the time course of the present experiment.
Section snippets
Oysters
One-year-old cupped Pacific oysters C. gigas were purchased in November 2008 from a shellfish farm located on the French Mediterranean coast. Animal size was around 40 mm in length, 5 g of total weight. Animals did not present any mortality or other symptom of disease prior the experiment. Moreover, no abnormal mortality event has been reported at the shellfish farm location in 2008. Oysters were placed in the Ifremer's facilities (Laboratoire de Génétique et Pathologie, La Tremblade, France) in
Oyster mortality
Batches of oysters infected by intramuscular injection of OsHV-1 suspensions were characterized by a sudden mortality (around 55%) occurring only 2 days after injection (Fig. 1). At Day 6 post-injection, the cumulative mortality was around 80% and reach 90% at the end of the experiment (Day 10).
Mortalities rates observed in batches of oysters cohabited with moribund oysters were significantly lower than those recorded in oysters infected by intramuscular injection (P < 0.001). Mortality rates
Discussion
Previous studies have led to hypothesize ways of OsHV-1 transmission in the Pacific oyster C. gigas. It has been notably suggested that asymptomatic OsHV-1 infected oysters may play a role of reservoirs of the virus and that seawater may act as a vector in the horizontal transmission of OsHV-1 (Arzul et al., 2002, Le Deuff et al., 1996). Although experimental OsHV-1 infections has been carried out successfully in C. gigas at larval stages (Le Deuff et al., 1994, Le Deuff et al., 1996), attempts
Acknowledgements
Authors wish to express our gratitude to M. Sallas from “CAT de Maguelone”, at Palavas for providing healthy animals. We also are grateful to P. Legall from Ifremer Sète station for all care given to oysters and shipment from Prévost lagoon, and to P. Haffner for his technical assistance in bacteriology.
References (47)
- et al.
French scallops: a new host for Ostreid herpesvirus-1
Virology
(2001) - et al.
Detection of oyster herpesvirus DNA and proteins in asymptomatic Crassostrea gigas adults
Virus Research
(2002) - et al.
Diagnosis of Ostreid herpesvirus 1 in fixed paraffin-embedded archival samples using PCR and in situ hybridisation
Journal of Virological Methods
(2004) - et al.
Allometric relationships and effects of temperature on clearance and oxygen consumption rates of Crassostrea gigas (Thunberg)
Aquaculture
(1995) - et al.
A herpes-like virus from the European oyster Ostrea edulis L.
Journal of Invertebrate Pathology
(1993) Evolution of the herpesviruses
Veterinary Microbiology
(2002)Ultrastructural and ultracytochemical observations on Bonamia sp. in oysters (Tiostrea chilensis), with a consideration of organelle function
Aquaculture
(1992)- et al.
Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix
Journal of Food Protection
(2004) - et al.
Herpes-like virus detection in infected Crassostrea gigas spat using DIG-labelled probes
Journal of Virological Methods
(2002) - et al.
Rapid and sensitive detection of Ostreid herpesvirus 1 in oyster samples by real-time PCR
Journal of Virological Methods
(2008)