Abstract:
Foodborne disease from consumption of shellfish is a significant public health concern, especially since rates of consumption continue to increase worldwide (Potasman, Paz et al. 2002). Although only 2000 cases of shellfish-associated illness were reported from 1991-1998 in the United States, the total number is estimated to be 4.5 million cases of illness per year (Mead and al 1999; Wallace, Guzewich et al. 1999; Shieh, Monroe et al. 2000). Despite widespread testing of shellfish for Vibrio species and their associated marine toxins, Noroviruses and Hepatitis A Virus actually pose the greatest threat to public health. (Koopmans, von Bonsdorff et al. 2002; Potasman, Paz et al 2002).
Washington State currently uses fecal coliform concentration in growing water to determine the safety of consuming shellfish harvested from Puget Sound (PS). The level of enteric pathogens and other indicator organisms in shellfish tissues are not routinely tested, and have not been adequately described in PS shellfish. The level of viral contamination of PS shellfish has never been studied, although outbreaks of shellfish-borne viral diseases have been reported in Washington State as a result of oyster consumption.
Current methods for testing shellfish tissues for viral contamination are cumbersome and inefficient, and few adequate direct-detection techniques are available. Real-Time Reverse Transcription PCR is swiftly becoming the method of choice for detecting virus in shellfish meat, but still has not been accepted for routine, large-scale testing. (Kageyama, Kojima et al. 2003; Nishida, Kimura et al. 2003; Kageyama, Shinohara et. al 2004; Formiga-Cruz, Hundesa et al. 2005; Jothikumar, Lowther et al. 2005). Resistance to using this method is a result of the tedious dissection and extraction procedures required for isolating viruses from the digestive tissues of oysters, and the lack of a standardized protocol (including primer/probe sets) for Real-Time Reverse Transcription PCR. (Bouchriti and Goyal 1993). Leading protocols are those described by DN Lees (1994) in which shellfish are homogenized, PEG precipitated and then centrifuged at 10,000xg and also by M Formiga-Cruz (2002), in which viruses are isolated from oyster homogenate in multiple centrifugation steps, starting at 2,170 x g for 15 min, then 39,800 x g for 45 min, and then ultracentrifuged at 81,584 x g for 1 h to pellet all viral particles. A third commonly followed protocol which required a high speed, but not ultracentrifuge, is that put forth by YC Sheih, (1999) in which viruses in the oyster homogenate are adsorbed into solids, centrifuged at 2,000 x g for 20 min, then eluted in glycine before being centrifuged again at 5,000 x g for 20 min, then PEG precipitated and spun at 6,700 x g for 30 min, followed by solvent extraction of resuspended viruses and a final PEG precipitation followed by centrifugation at 14,000 x g for 15 min.
Two modifications were made to the current methods to overcome the problems of labor-intensive procedures and the need for a high-speed centrifuge. The first was ultra-cold freezing of oyster tissue prior to dissection, and the second was direct RNA isolation form oyster lysate. Since shellfish tissue may contain high levels of PCR inhibitors, and because viruses and other pathogens collect in the digestive diverticulum (DD) of shellfish, recently described methods have relied on isolation of the digestive tissues prior to viral analysis in effort to reduce inhibition. Pre-freezing the intact oyster resulted in separation of the adductor muscles from the shell and made it significantly easier to remove oysters from their shells. More importantly, freezing allowed for easier location and dissection of the DD within the organism. Isolating RNA directly from the oyster lysate rather than relying on an adsorption/elution technique increases the sensitivity of the assay. Chloroform extraction removed the majority of particulate matter from the oyster lysate, making near-constant reload of Qiagen RNeasy columns possible. Because the amount of virus in shellfish tissue is commonly small, being able to sample a larger volume is critical in order to capture the small amount of virus which may be present. To ensure wide breadth of detection, two sets of broadly reactive primers and Taqman probes were used in the real time RT-PCR assay to detect the viral RNA captured on the Qiagen columns. These modifications resulted in a method for detection of virus in oyster tissue that significantly reduced both personnel time required to complete the assay and time to detection of virus in the samples.