PHYSICAL INTERVENTIONS

Historically, one of the oldest processing prac-tices for molluscan shellfi sh sanitation was depura-tion, which has been practiced throughout the world for approximately 100 years (Canzonier, 1991). This application was driven by a series of typhoid fever outbreaks during the 1920s that were associated with the consumption of raw oysters (Canzonier, 1991) and led to the establishment of the National Shellfi sh Sanitation Program (NSSP). The process involves the removal of potential pathogens by placement of shell-fi sh in sanitized seawater that is usually treated either by ozonation (Schneider et al., 1991) or by UV light (Tamplin and Capers, 1992) during recirculation into wet storage tanks (Furfari, 1991). UV light irradia-tion is currently the most common depurairradia-tion method in the United States and the United Kingdom. UV light allows continuous dosage, and numerous stud-ies have shown that it is effective in inactivating bac-teria (Cabelli and Haffernau, 1971; Cortelyou et al., 1954; Kelly, 1961; Kawabata and Harada, 1959) and viruses (Hill et al., 1969, 1970). The bactericidal activity disrupts unsaturated bonds, particularly purine and pyrimidines found in DNA, and lethal changes are directly related to the depth of penetra-tion and amount of particulate matter (Huff et al., 1965). Depuration is the preferred method for removal of fecal coliforms, enteric bacteria, and viral pathogens in many countries and has a distinct advan-tage over other treatments in that it does not kill mol-lusks (Richards, 1990). Unfortunately, the depuration process does not effectively remove Vibrio species from shellfi sh (Vasconcelos and Lee, 1972; Tripp, 1960; Colwell and Liston, 1960), as vibrios persisted in shellfi sh for up to 96 hours of treatment at concen-trations that were similar to those in untreated shell-fi sh (Rodrick and Schneider, 1991). Thus, vibrios appear to be fi rmly attached to the shellfi sh tissues and are not effectively removed by depuration.

In 2003 the FDA and the NSSP set forth guide-lines intended to minimize consumer risk from natu-rally occurring V. vulnifi cus in oysters. The guidance document focused on consumer education, harvest restrictions, and postharvest treatments (PHP) to ensure product safety. The oyster industry responded to these guidelines by implementing pre- and posthar-vest controls aimed at reducing levels of V. vulnifi cus and V. parahaemolyticus. Control methods have been evaluated and adjusted in an effort to reduce annual vibrio infections. Implementation of these methods requires validation and verifi cation that these treat-ments meet standards for reduction of vibrios in Consequently, strict time and temperature

regula-tions have been established to limit exposure of sea-food to elevated temperatures that will promote the growth of these species (NSSP, 2005). Vibrio patho-gens die or become nonculturable at refrigeration temperatures. Experimental evidence indicates that Vibrio species grown at refrigeration temperatures remain viable but cannot be recovered by standard culture methods. Entry into a dormant “viable but nonculturable” state has been postulated (Xu et al., 1982; Oliver et al., 1991). Furthermore, viable but nonculturable bacteria retain virulence in animal models but with increased infectious dose (Oliver and Bockian, 1995). Vibrios are also sensitive to elevated temperatures, with upper limits for growth temperature in V. parahaemolyticus and V. vulnifi -cus at 44 and 42C, respectively. However, these bacteria die at even mildly elevated temperatures (45 to 55C), and infections are very rarely associated with properly cooked seafood. Beuchat (1973) reported thermal resistance at 47C (D47 values) ranging from 0.8 to 65.1 minutes, but heat resis-tance increased at higher inocula and with addition of 7% NaCl. Comparison of heat resistances for pathogenic vibrios showed that D55 values varied among species (Johnston and Brown, 2002). V. para-haemolyticus (D55 of 1.75 min) was considerably more resistant compared to V. vulnifi cus and V. chol-erae (12 and 22.5 s, respectively). However, stan-dard pasteurization conditions (70C for 2 min) produced a 7-log reduction in all three species.

Some food processing, such as mild steaming, may not thoroughly heat the entire oyster and result in survival of vibrios postcooking.

Vibrios are fairly tolerant of high pH but will not grow below pH 6 (Beuchat, 1973). Decimal reduction time was reduced signifi cantly at 53C for pH 5 to 6 compared to pH 7. The primary enrich-ment medium (alkaline peptone water) uses pH 8 to achieve selective growth of these species. Survival and growth of Vibrio species in seafood are also related to salinity. All vibrios require some salt for growth, but species differ greatly in their responses to salinity. For example, V. parahaemolyticus and V. vulnifi cus are both moderate halophiles and will not grow below 0.1% NaCl. However, V. parahaemolyticus grows well at 8% NaCl, while V. vulnifi cus does not survive at 8.5% and growth is inhibited at 6.5% NaCl (Kelly, 1982). In contrast, V. cholerae will grow in culture medium without additional salt, and V. alginolyticus is differentiated from the other pathogens by the abil-ity to grow in 10% NaCl. Survival of V. parahaemo-lyticus at lower temperatures can increase with addition of NaCl or by adjusting the pH of growth medium (Beuchat, 1973).

prolonged exposure to low temperature storage, vibrios can also become nondetectable on standard media but still be viable and in virulence (Baffone et al., 2003).

Ultra-Low Temperature Treatment

Recently, ultra-low temperature treatment (70C) has been shown to effectively reduce vibrios when it was followed by extended frozen stor-age at 20ºC for 1 to 2 weeks, depending on the process. Ultra-low freezing can be achieved by immer-sion of shellstock in liquid nitrogen or CO₂, and liquid nitrogen treatment was recently validated as an oyster PHP for reduction of V. vulnifi cus. Vibrio content in oysters following nitrogen treatment and frozen storage was signifi cantly reduced to levels that were below those specifi ed by the NSSP (Wright et al., 2007). This treatment has the advantage of produc-ing a high-quality product with extended shelf life.

Heat Treatment

Heating is also an effective treatment for elimi-nation of Vibrio species, as evidenced by the lack of disease association with cooked seafood products.

All Vibrio species die rapidly at temperatures exceeding 55C (Johnston and Brown, 2002). Heat shock treatment involves immersion in hot water (50ºC for 5 to 10 minutes) combined with frozen storage and achieves the desired reduction within 2 weeks (Andrews et al., 2000). Traditional pasteur-ization is also done at 75C for 8 min and can reduce vibrio counts to safe levels for the consumer but pro-duces some biochemical and sensory changes in the meats (Cruz-Romero et al., 2007). Heat treatment of oysters also improves shucking yields compared to untreated oysters and benefi ts both the consumer and the distributor.

HPP

Recent progress in the high-pressure processing (HPP) of foods in general, and oysters in particular, suggests that this method can be applied to shellfi sh.

Application of elevated pressure dramatically reduces the numbers of Vibrio species, and produces a “self-shucking” product. Treatment of oysters at 400 MPa for 10 minutes resulted in an about 5-log-unit reduc-tion of total viable count, H₂S-producing organisms, lactic acid bacteria, and coliforms (Lopez-Caballero et al., 2000). The use of HPP at 310 MPa caused the opening of the shell with 100% effi ciency as the adductor muscle of the oyster was detached in the process (He et al., 2002). Recent studies have shown the ability of freezing coupled with frozen storage as a means of reducing the number of recoverable oysters. Approved and validated treatments include

high hydrostatic pressure, pasteurization (heat shock), and individual quick freezing. These treatments are generally used in combination with approved trans-port and storage practices involving icing, refrigera-tion, and/or freezing.

Refrigeration

Seafood held at ambient temperatures can sup-port rapid growth of Vibrio species (Gooch et al., 2002; Cook, 1994); therefore, both warm and cold temperature processing have been used in controlling and killing pathogenic Vibrio species in molluscan shellstock. The simplest and most common process-ing practice for all fi sh and shellfi sh handlprocess-ing is the immediate transfer of the product to ice or refriger-ated storage. Refrigeration of oysters is required within 10 hours of harvest in summer months; how-ever, the time-temperature matrix varies with the ambient air temperature and infection risk. In shell-fi sh areas with conshell-fi rmed association with a vibrio outbreak, the allowable time between harvest and refrigeration is reduced. Bacterial levels decline some-what with refrigeration, but vibrios are not elimi-nated in oysters, even with an extended exposure of 10 to 15 days (Kaspar and Tamplin, 1993; Cook, 1994; Murphy and Oliver, 1992). The rate of decline is inoculum dependent, and survival at low tempera-ture is also enhanced by more gradual rates of cool-ing (Bryan et al., 1999). Furthermore, growth was observed with food products at 9.5 to 10C. Tem-perature controls are even more relevant for intertidal oysters, as these oysters are exposed to ambient air temperatures and may warm to levels that promote growth of Vibrio species. Therefore, harvesting dur-ing intertidal exposure is avoided, or oysters are transferred to deeper, cooler seawater prior to har-vest. Maintenance of refrigerated or freezing temper-atures during transport and storage is also regulated, and lack of compliance with these recommendations may account for a signifi cant proportion of disease.

Unfortunately, refrigerated storage results in only moderate bacterial reductions and is not appro-priate for PHP that is aimed at risk reduction. Immer-sion of oysters in ice prior to refrigeration results in greater reduction compared to simple refrigeration but also does not completely eliminate V. vulnifi cus in oysters (Quevedo et al., 2005). Furthermore, this study also found that ice immersion could increase the number of fecal coliforms in oysters, suggesting that this method should be used with caution. Thus, product with a signifi cant bacterial load at harvest will still present increased risk to the consumer, even following rapid decreases in temperature. After

biological controls use microbiological products or microorganisms as food additives to reduce pathogen load. These “benefi cial” bacteria can outcompete pathogens, limit their growth, or be lethal to poten-tial pathogens. Antibiotics are generally not added to foods, but an exception to this rule is a class of anti-microbial molecules, termed bacteriocins, that are produced by bacteria and are specifi cally lethal to other bacteria. Applications are primarily against gram-positive bacteria (Lipinska, 1977), and the bacteriocin nisin is now permitted in over 40 coun-tries worldwide, including the United Kingdom, the European Union, Russia, and India (http://www.

foodnavigator.com). Acceptance of these molecules as biological controls is a consequence of their natu-ral presence in milk products, lack of activity against higher organisms, and demonstration of effective-ness toward Listeria monocytogenes (Farber, 1993).

Unfortunately, currently accepted bacteriocins are not effective against gram-negative bacteria and thus will not clear pathogenic vibrios in oysters.

Government Regulation

Standard mitigation and monitoring strategies for shellfi sh were originally based on fecal coliform content, but this type of analysis is ineffective in pre-venting naturally occurring contamination of vibrios in shellfi sh. Warning labels on oyster products and extensive educational programs targeted at-risk indi-viduals with underlying diseases, such as cirrhosis, hemochromatosis (iron overload), diabetes, or immune system dysfunction. Unfortunately, these strategies did not completely eliminate disease mor-tality from V. vulnifi cus diseases (NSSP, 2003), and a small number of cases are still reported annually, presumably because refrigerated temperature guide-lines limit growth but do not eliminate vibrios in oysters. Furthermore, problems with seafood safety have been exacerbated by recent outbreaks of V. parahaemolyticus. PHP of oysters to reduce the numbers of vibrios has been mandated for Gulf Coast oysters. PHP is now implemented for 25%

of oyster shellstock from the Gulf Coast and requires a minimum reduction of 3.52 log most probable numbers (MPN)/g reduction in vibrio content in order to validate a treatment for commercial pro-cessing. California also requires all oysters harvested from Gulf Coast states to be treated during certain months. Increased incidence of V. parahaemolyticus disease necessitated expanding PHP programs to include Pacifi c Coast oysters, and new guidelines provide for monitoring oysters with recommended closures when V. parahaemolyticus levels exceeded 10,000 bacteria/g.

V. vulnifi cus bacteria in Gulf Coast oysters (Parker et al., 1994).

Irradiation

The United States has lagged behind other coun-tries in the application of food irradiation technolo-gies partially due to the U.S. Food, Drug, and Cosmetic Act of 1958, which classifi ed irradiation as a food additive. In the 1980s, the FDA approved irradiation as a safe and effective method of decreasing or elimi-nating harmful bacteria in herbs, spices and vegetable seasoning, fruits, vegetables, and grains (Morrison, 1986). Meat, poultry, and other foods, including oys-ters, were not approved by the FDA until 2006. Oys-ters appear tolerant to irradiation processing at levels of a 2.5-kGy absorbed dose, as normal shelf-life is maintained with no increase in mortality compared with untreated control oysters (Mallett et al., 1987).

However, higher levels of irradiation increased mor-tality and resulted in a yellow exudate and an unpalat-able product. Furthermore, V. parahaemolyticus cultures were less sensitive to irradiation compared to V. vulnifi cus.

Relaying

The basic concept of relaying involves transfer-ring shellfi sh from restricted, polluted waters to areas approved for harvesting and permitting the process of natural biological purifi cation. Filter-feeding shell-fi sh can purge themselves of certain abiotic and biotic substances when transferred or “relayed” to rela-tively clean seawater. The rate of purging depends on the type of contaminant, species of shellfi sh, water quality, length and method of relay, and various envi-ronmental factors. Erdman and Tennant (1956) showed an average 85% reduction in the number of coliform bacteria in the soft-shell clam (Mya are-naria). Relaying also includes transporting oysters to areas with higher salinity in order to reduce levels of V. vulnifi cus (Motes et al., 1998). Problems associ-ated with relaying are that it is a labor-intensive pro-cess and that up to 50% of the original harvest can be lost (Furfari, 1979). Unfortunately, depuration and relaying to higher salinities present logistical and reg-ulatory problems and could increase contamination by other more halophilic Vibrio species.

Food Additives and Biological Controls Although consumer markets for the treated sea-food have been growing, a substantial number of shellfi sh consumers still prefer live, fresh, raw oysters.

Various food additives have also been investigated but were not effective in live oysters (Sun and Oliver, 1994; Birkenhauer and Oliver, 2003). Probiotics or

Species Identifi cation

Commercial formats, such as the API 20E and Biolog systems, are available for biochemical identifi -cation of vibrios. However, these assays are costly, and the phenotypic plasticity of these species frequently complicates their application. Antibodies for species-, serotype-, and toxin-specifi c identifi cation are also available but rely upon expressed antigen and may require cell processing for detection. Lipo polysaccharide antibody or “O” antigen has been used traditionally to identify and classify epidemic V. cholerae. Clinical strains V. vulnifi cus and V. parahaemolyticus show more heterogeneous serotypes, which are not useful for species identifi cation. Increasingly, molecular meth-ods are used for species identifi cation because they improve specifi city and sensitivity of detection. A non-radioactive DNA probe format, using alkaline phos-phatase-labeled oligonucleotides, is the FDA-specifi ed method for vibrio identifi cation (Kaysner and DePaola, 2004). DNA probes for V. cholerae recognize genes for CT (Wright et al., 1992), and speciesspecifi c identifi -cation of V. parahaemolyticus targets the gene for a thermal-labile hemolysin, while potentially pathogenic isolates are discriminated by probes detecting genes for TDH and TRH virulence-associated hemolysins, described above (Nordstrom et al., 2006). Probes for V. vulnifi cus are based upon a cytolysin gene, vvhA (Wright et al., 1993).

A number of PCR methods are also used to amplify species- or virulence-specifi c DNA (Brauns et al., 1991), and more recent methods include real-time detection and quantitative PCR (Panicker et al., 2004; Panicker and Bej, 2005; Campbell and Wright, 2003; Tarr et al., 2007; Blackstone et al., 2003; Lyon, 2001). Multiplex PCR analyses for simultaneous iden-tifi cation of multiple food-borne pathogens, including vibrios, have been reported (Wang et al., 1997; Lee et al., 2003; Brasher et al., 1998). Unfortunately, PCR reactions are often inhibited by food matrices and generally require strain isolation and some purifi ca-tion of the DNA template for optimum sensitivity.

Improved separation technologies may function to concentrate and increase sample size, while removing inhibitors (Stevens and Jaykus, 2004).

Enumeration of Pathogenic Vibrios

The Food and Drug Administration (FDA) Bac-teriological Analytical Manual (http://www.cfsan.

fda.gov/~ebam/bam-9.html) specifi es two basic ap-proaches for enumeration of vibrios: (i) MPN and (ii) colony counts determined by direct spread plating using DNA probe identifi cation of colonies (Kaysner and DePaola, 2004). Enumeration of vibrios generally begins with homogenization of fi sh or shellfi sh tissue DETECTION METHODS FOR CONFIRMATION

AND TRACE-BACK OF CONTAMINATED