166
D. Polo et al.
Traditional bacterial indicators of fecal contamination, on which sanitary
controls are based, and enteric viruses significantly differ in terms of transmission,
resistance to sewage treatment, and persistence in the environment (Da Silva et al.
2007). Depuration allows the purging of gastrointestinal contents under controlled
conditions in order to reduce the likelihood of transmitting infectious pathogens.
The efficacy of depuration in the elimination of such agents is a critical issue for
the development of improved shellfish sanitary controls. Although depuration can
reduce bacterial levels from shellfish, the removal of viral particles is not as effective
(Ueki et al. 2007) and therefore, depuration may not ensure the absence of viral
contamination (Croci et al. 2007; Loisy et al. 2005; Schwabm et al. 1998). Periodic
outbreaks of enteric diseases linked to shellfish which comply with legal standards
and/or are subjected to depuration, indicate the inability of both bacterial indicators
(Romalde et al. 2002; Umesha et al. 2008) and commercial depuration (Chalmers
and McMillan1995; Heller et al. 1986; Le Guyader et al. 2003, 2006a) to predict
the viral risk.
Depuration is a complex biological process, which varies according to bivalve
species, pathogens (bacteria or virus) and possibly between viral species. Therefore,
a better understanding of specific behavior of diverse viruses in different bivalve
species is needed. Experimental depuration systems provide a useful tool to find
the best operational parameters and to improve commercial depuration. This study
evaluates and compares by reverse transcription-real time RT-PCR (qRT-PCR) the
effectiveness of depuration in clams and mussels subjected to bioaccumulation with
murine norovirus (MNV-1), as a surrogate of human norovirus (Wang et al. 2008)
in an experimental depuration system.
Materials and Methods
Sample Collection and Depuration Facilities
A closed experimental depuration system (isothermal ASE M BINS system, 500 kg
of capacity) (AdriaticSeaAquarium and Equipment SRL, San Clemente, Italy) with
mechanical, biological and chemical static filter systems, thermal control and water
sterilization by ozone and UV-C radiation was employed (Fig. 14.1). Ten depuration
experiments were carried out with Manila clams (Venerupis philippinarum) and
mussels (Mytilus galloprovincialis) (five with each species) after bioaccumulation
with MNV-1. Each experiment was performed with 60 kg of mollusc.
Bioaccumulation of Virus by Molluscs
Molluscs were obtained from local producers, kept at 4
ı C during shipment and
arrived at the laboratory within the next 4 h. After 24 h of acclimatization in tanks
with 100 l of seawater and continuous aeration, bioaccumulation was performed
D. Polo et al.
Traditional bacterial indicators of fecal contamination, on which sanitary
controls are based, and enteric viruses significantly differ in terms of transmission,
resistance to sewage treatment, and persistence in the environment (Da Silva et al.
2007). Depuration allows the purging of gastrointestinal contents under controlled
conditions in order to reduce the likelihood of transmitting infectious pathogens.
The efficacy of depuration in the elimination of such agents is a critical issue for
the development of improved shellfish sanitary controls. Although depuration can
reduce bacterial levels from shellfish, the removal of viral particles is not as effective
(Ueki et al. 2007) and therefore, depuration may not ensure the absence of viral
contamination (Croci et al. 2007; Loisy et al. 2005; Schwabm et al. 1998). Periodic
outbreaks of enteric diseases linked to shellfish which comply with legal standards
and/or are subjected to depuration, indicate the inability of both bacterial indicators
(Romalde et al. 2002; Umesha et al. 2008) and commercial depuration (Chalmers
and McMillan1995; Heller et al. 1986; Le Guyader et al. 2003, 2006a) to predict
the viral risk.
Depuration is a complex biological process, which varies according to bivalve
species, pathogens (bacteria or virus) and possibly between viral species. Therefore,
a better understanding of specific behavior of diverse viruses in different bivalve
species is needed. Experimental depuration systems provide a useful tool to find
the best operational parameters and to improve commercial depuration. This study
evaluates and compares by reverse transcription-real time RT-PCR (qRT-PCR) the
effectiveness of depuration in clams and mussels subjected to bioaccumulation with
murine norovirus (MNV-1), as a surrogate of human norovirus (Wang et al. 2008)
in an experimental depuration system.
Materials and Methods
Sample Collection and Depuration Facilities
A closed experimental depuration system (isothermal ASE M BINS system, 500 kg
of capacity) (AdriaticSeaAquarium and Equipment SRL, San Clemente, Italy) with
mechanical, biological and chemical static filter systems, thermal control and water
sterilization by ozone and UV-C radiation was employed (Fig. 14.1). Ten depuration
experiments were carried out with Manila clams (Venerupis philippinarum) and
mussels (Mytilus galloprovincialis) (five with each species) after bioaccumulation
with MNV-1. Each experiment was performed with 60 kg of mollusc.
Bioaccumulation of Virus by Molluscs
Molluscs were obtained from local producers, kept at 4
ı C during shipment and
arrived at the laboratory within the next 4 h. After 24 h of acclimatization in tanks
with 100 l of seawater and continuous aeration, bioaccumulation was performed