High Pressure Inactivation of HAV Within Oysters: Comparison of Shucked Oysters with Whole-In-Shell Meats
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- Kingsley, D.H., Calci, K., Holliman, S. et al. Food Environ Virol (2009) 1: 137. doi:10.1007/s12560-009-9018-5
High pressure inactivation of hepatitis A virus (HAV) within oysters bioaccumulated under simulated natural conditions to levels >105 PFU/oyster has been evaluated. Five minute treatments at 20°C were administered at 350, 375, and 400 MegaPascals (MPa). Shucked and whole-in-shell oysters were directly compared to determine if there were any differences in inactivation levels. For whole-in-shell oysters and shucked oysters, average values obtained were 2.56 and 2.96 log10 inactivation of HAV, respectively, after a 400-MPa treatment. Results indicate that there is no significant inactivation difference (P = 0.05) between inactivation for whole-in-shell oysters as compared to shucked oysters observed for all pressure treatments. This study indicates that commercial high pressure processing applied to whole-in-shell oysters will be capable of inactivating HAV pathogens.
KeywordsHAVHigh pressureShell oysters
Bivalve shellfish are efficient concentrators of viral pathogens in the water column and can pose health risks to raw shellfish consumers. High pressure processing (HPP) has emerged as a valuable intervention technology for the shellfish industry. Consumers demand uncooked shellfish, and this non-thermal technology retains much of the raw taste and character. For commercial shellfish, HPP has several utilities. HPP facilitates “shucking”, or separation of the meat from the shell, resulting in plump and intact bivalve meat. Also HPP is an approved technology for inactivation of Vibrio vulnificus (Vv), a warm water estuarine bacteria which is highly pathogenic for susceptible populations. Ninety-five per cent of the deaths associated with seafood consumption in the US annually are caused by Vibrio vulnificus (Oliver and Kaper 2001). Research evaluating the utility of HPP for inactivation of other shellfish-borne pathogens indicates excellent prospects for commercial application. For example several isolates of Vibrio parahemolyticus, an estuarine bacteria associated with bacterial gastroenteritis, are inactivated by high pressures only approximately 25 MPa higher than those used for Vv (Cook 2003; Kural et al. 2008), and HPP is also effective against Vibrio cholera (Berlin et al. 1999).
Viruses of fecal origin, such as human norovirus and hepatitis A virus (HAV), represent a unique challenge to the shellfish industry. Once shellfish ingest viruses from the water column, the infectious viruses are efficiently retained within bivalves for extended periods of time (Kingsley and Richards 2003; Loisy et al. 2005). Other than cooking shellfish, there are limited options for rendering them infectious virus free. However, recent study applying high pressure processing suggests that HPP has excellent prospects for inactivating these viruses. Human norovirus is currently very difficult to propagate in vitro (Straub et al. 2007). Consequently, research surrogates must be used to evaluate HPP potential to inactivate norovirus. Feline calicivirus (FCV) is very sensitive to high pressure application with a 5 min 275-MPa treatment completely inactivating 107 TCID50/ml (Chen et al. 2005; Kingsley et al. 2002). A more recently discovered, phylogenetically closer surrogate, murine norovirus (Wobus et al. 2006) is somewhat more resistant to pressure than FCV. However, a 5-min 400-MPa treatment at 5°C was sufficient to inactivate 4.05 log10 PFU within oyster tissues (Kingsley et al. 2007). It has been shown that HAV can be inactivated directly within contaminated shellfish (Calci et al. 2005) with log10 reductions averaging 1.28, 2.32, and 3.15 observed for 350, 375, and 400 MPa treatments, respectively, performed for 1 min at 9°C.
All research to date has been performed with either virus stocks or shucked virus-contaminated oyster meats. Commercial application of HPP is performed on whole-in-shell oysters; therefore it is desirable to determine if there are any important differences between HAV inactivation observed for whole-in-shell as compared to shucked oysters. A priori, one would not expect any differences between whole and shucked oysters since HPP is an instantaneous process in which pressure is uniformly applied throughout the sample and bivalve shells cannot form a pressure tight seal. However, it is known that different food matrices may have variable heats of compression. Furthermore, the temperature at which pressure is applied can significantly influence virus inactivation (Chen et al. 2005; Kingsley et al. 2006, 2007). While the presence of a shell should not in any way diminish the pressure experienced by the oyster meat, the shell could conceivably result in a non-uniform, or at least different, adiabatic heat transfer that could influence virus inactivation. In this publication, we confirm that there is essentially no significant difference between HAV inactivation observed in-shell and inactivation observed in shucked oyster meats.
Materials and Methods
Virus and Oysters
HAV was obtained from the American Type Culture Collection (Manassas, VA) as VR1402, a cell culture-adapted cytopathic clone of strain HM-175/18f. HAV was propagated in fetal rhesus monkey kidney (FRhK-4) cells in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen Corp. Carlsbad, CA). All viral stocks were stored at −70°C in Dulbecco’s Modified Eagle Medium (DMEM; Gibco-BRL, Gaithersburg, MD) with 10% fetal bovine serum prior to use. Eastern oysters (Crassostrea virginica) were harvested from an approved area in Mobile Bay, AL. Commercial-sized oysters were placed into a depuration flume at the US FDA Gulf Coast Seafood Laboratory, Dauphin Island, AL. Oysters were maintained for >3 week prior to being transferred to a flume which utilized single-pass UV-treated natural seawater. Salinities ranged from 5 to 20 parts per thousand (ppt). Oysters were contaminated with HAV in an accumulation tank as described previously (Calci et al. 2005) with each oyster being exposed to approximately 5 × 107 PFU of HAV.
High Pressure Treatment
Whole-in-shell oysters and shucked samples were triple bagged in 6 × 8 inch, 3 mil STD Barrier Nylon/PE vacuum pouches (Prime Source, Inc.). Bags were heat-sealed using an Impulse Food Sealer (American International Electric Co., Whittier, CA) according to the manufacturer’s instructions. Pressurization of oyster samples was performed for 5 min using a Quintus 35-L food press (QFP 35L-600; Avure technologies Inc., Kent, WA). Samples were pressurized at 350, 375, and 400 MPa for 5-min at ~17–22°C. Come-up times to reach final pressures was approximately 90 s for 350–400 MPa treatments. Pressure-release time was <3 s. After processing, the refrigerated samples were shipped on ice overnight to the USDA Microbial Food Safety Research Unit at Dover, DE for virus extraction and assay.
Virus Extraction and Plaque Assays
Virus-contaminated shellfish (three shellfish per group) were removed from pressurized sealed pouches and placed in 50-ml conical tubes and briefly centrifuged in a table top centrifuge to facilitate separation of oyster meat from oyster liquor. Non-pressurized (0 MPa) HAV-contaminated positive controls were also tested. Virus extractions were performed as described by Calci et al. (2005). Two ml of extract or 2 ml of 10-fold serial dilutions were made in Earle’s balanced salt solution and plaque assays were performed in triplicate using FRhK-4 cells as described by Richards and Watson (2001). The detection limit for the cell culture assay was approximately 33 PFU/oyster group or 1.5 log10, and 6 ml of the original 200 ml of extract was assayed for infectious virus.
Three independent trials were conducted for all pressure treatments. The data were analyzed using Excel software (Microsoft). Statistical analyses were performed using Sigma Stat 3.5 (Systat Software, Inc., Chicago IL). A t-test was used to compare significant differences (P < 0.05) between treatments. A Q test was used to determine statistical outliers.
Results and Discussion
Viable HAV detected after HPP treatment of shucked and whole-in-shell oysters
Pressure applied (MPa)
Average log10 PFU (SE)
Previous results confirmed the potential effectiveness of HPP against HAV in cell culture media (Kingsley et al. 2002) and within shucked oyster meats after simulated natural bioconcentration (Calci et al. 2005). In this study, we demonstrated that HPP is also effective for whole-in-shell oysters and that the presence of a shell does not adversely influence inactivation of HAV within oysters. In the previous study (Calci et al. 2005), shucked oysters were treated over pressures ranging from 300 to 400 MPa for 1 min at 9°C. In this study, oysters were treated at 17–22°C for 5 min at pressures of 350, 375, and 400 MPa. Although pressure holding times and the initial temperature at which pressure was applied were different, results obtained were comparable to the previous study (Calci et al. 2005) with approximately 2.5 and 3 log10 PFU inactivation observed at 350 and 400 MPa, respectively. Curiously in this study, greater HAV inactivation at 350 MPa was observed with log10 reductions of 2.18 shucked and 2.26 for in-shell oysters, as compared to log10 reduction of 1.28 for the previous study (Calci et al. 2005). Whether this discrepancy reflects differences in temperature or treatment times or some other variable is unknown. It is known that the initial temperature at which pressure is applied can make a substantial difference in virus inactivation (Chen et al. 2005; Kingsley et al. 2006, 2007; Kingsley and Chen 2009). For HAV, colder temperatures result in substantially less inactivation of virus stocks in DMEM culture media (Kingsley et al. 2006). However, another study (Kingsley and Chen 2009) showed that for inactivation of HAV within an oyster matrix, initial sample temperature does not substantially influence the degree of inactivation observed. In total, this study validates the potential to inactivate HAV in a commercial setting and indicates that the presence of the bivalve shell during commercial bivalve processing will not have any adverse effects on the use of HPP for potential mitigation HAV.