ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 24 (2007) 549–558 www.elsevier.com/locate/fm
Review
Vibrio parahaemolyticus: A concern of seafood safety Yi-Cheng Sua,, Chengchu Liub a
OSU Seafood Laboratory, Oregon State University, 2001 Marine Drive, Room 253, Astoria, OR 97103, USA College of Food Science and Technology, Shanghai Fisheries University, 334 Jungong Road, Shanghai 200090, China
b
Received 17 July 2006; received in revised form 9 January 2007; accepted 11 January 2007 Available online 30 January 2007
Abstract Vibrio parahaemolyticus is a human pathogen that is widely distributed in the marine environments. This organism is frequently isolated from a variety of raw seafoods, particularly shellfish. Consumption of raw or undercooked seafood contaminated with V. parahaemolyticus may lead to development of acute gastroenteritis characterized by diarrhea, headache, vomiting, nausea, and abdominal cramps. This pathogen is a common cause of foodborne illnesses in many Asian countries, including China, Japan and Taiwan, and is recognized as the leading cause of human gastroenteritis associated with seafood consumption in the United States. This review gives an overview of V. parahaemolyticus food poisoning and provides information on recent development in methods for detecting V. parahaemolyticus and strategies for reducing risk of V. parahaemolyticus infections associated with seafood consumption. r 2007 Elsevier Ltd. All rights reserved. Keywords: Vibrio parahaemolyticus; Foodborne pathogen; Food poisoning; Shellfish; Detection methods; Seafood safety
Contents 1. 2.
3.
4. 5. 6.
7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Occurrence of Vibrio parahaemolyticus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Distribution in marine environments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Prevalence in shellfish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Incidence of Vibrio parahaemolyticus food poisoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Incidence in Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Incidence in Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Incidence in the United States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Virulence factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emerging strain of Vibrio parahaemolyticus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection of Vibrio parahaemolyticus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Most probable number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Polymerase chain reaction (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. DNA hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Chromogenic medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Controls and prevention. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Relaying and depuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Thermal processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. High-pressure processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: +1 503 325 4531; fax: +1 503 325 2753.
E-mail address:
[email protected] (Y.-C. Su). 0740-0020/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2007.01.005
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8.
7.5. Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Vibrio parahaemolyticus is a Gram-negative, halophilic asporogenous rod that is straight or has a single, rigid curve. It has a single polar flagellum and is motile when grown in liquid medium (Baumann and Schubert, 1984). This bacterium is a human pathogen that occurs naturally in the marine environments and frequently isolated from a variety of seafoods including codfish, sardine, mackerel, flounder, clam, octopus, shrimp, crab, lobster, crawfish, scallop and oyster (Liston, 1990). Consumption of raw or undercooked seafood, particularly shellfish, contaminated with V. parahaemolyticus may lead to development of acute gastroenteritis characterized by diarrhea, headache, vomiting, nausea, abdominal cramps and low fever. This bacterium is recognized as the leading cause of human gastroenteritis associated with seafood consumption in the United States and an important seafood-borne pathogen throughout the world (Kaysner and DePaola, 2001). Although the gastroenteritis caused by V. parahaemolyticus infection is often self-limited, the infection may cause septicemia that is life-threatening to people having underlying medical conditions such as liver disease or immune disorders. Two deaths were reported among three cases of wound infections caused by V. parahaemolyticus in Louisiana and Mississippi after Hurricane Katrina in 2005 (CDC, 2005). This article was prepared to provide an overview of V. parahaemolyticus foodborne illness and information on recent development in methods for V. parahaemolyticus detection and strategies for reducing risk of V. parahaemolyticus infections associated with seafood consumption.
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below 16 1C (DePaola et al., 1990). However, the densities of V. parahaemolyticus in seawater could increase to 1000 cell/100 ml in the water when water temperatures increased to around 25 1C (DePaola et al., 1990; Kaneko and Colwell, 1973). A recent study of occurrence of V. parahaemolyticus in Oregon oyster-growing environments between November 2002 and October 2003 also found a positive correlation between V. parahaemolyticus in seawater and water temperatures with the highest populations of V. parahaemolyticus in water being detected in the summer months (Duan and Su, 2005a). 2.2. Prevalence in shellfish
2. Occurrence of Vibrio parahaemolyticus
The degree of V. parahaemolyticus contamination in raw shellfish is also known to relate to the water temperatures. Therefore, it is more likely to detect V. parahaemolyticus in oysters harvested in the spring and the summer than in the winter. Although the density of V. parahaemolyticus in contaminated oysters is usually lower than 103 cfu/g at harvest (Kaysner and DePaola, 2000), it could exceed 103 cfu/g in oysters harvested from warmer seawater (DePaola et al., 2000) and the organism can multiply rapidly in oysters upon exposure of elevated temperatures. Studies have shown that populations of V. parahaemolyticus in unrefrigerated oysters could increase rapidly to 50–790 folds of its original level within 24 h of harvest if oysters were exposed to 26 1C (Gooch et al., 2002). A survey of 370 lots of oysters sampled from restaurants, oyster bars, retail and wholesale seafood markets throughout the US between June 1998 and July 1999 found a seasonal distribution of V. parahaemolyticus in market oysters with high densities (some exceeded 1000 MPN/g) being detected in the summer months (Cook et al., 2002).
2.1. Distribution in marine environments
3. Incidence of Vibrio parahaemolyticus food poisoning
The distribution of V. parahaemolyticus in the marine environments is known to relate to the water temperatures. Studies have shown that the organism was rarely detected in seawater until water temperatures rose to 15 1C or higher. Ecological study of V. parahaemolyticus in the Chesapeake Bay of Maryland found that V. parahaemolyticus survived in sediment during the winter and was released from sediment into water column when water temperatures rose to 14 1C in late spring or early summer (Kaneko and Colwell, 1973). Another survey of nine U.S. coastal states conducted between 1984 and 1985 reported an average low density of V. parahaemolyticus (4 cell/ 100 ml) in seawater when water temperatures dropped
3.1. Incidence in Asia V. parahaemolyticus was first recognized as a cause of food-borne illness in Osaka, Japan in 1951 (Daniels et al., 2000b). It caused a major outbreak of 272 illnesses and 20 deaths associated with consumption of sardines. Since then, V. parahaemolyticus has been reported to account for 20–30% of food poisoning cases in Japan (Alam et al., 2002) and identified as a common cause of seafood-borne illness in many Asian countries (Wong et al., 2000; Chen et al., 1991; Deepanjali et al., 2005). V. parahaemolyticus was the leading cause of food poisoning (1710 incidents, 24,373 cases) in Japan between 1996 and 1998 (IDSC,
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1999) and accounted for 69% (1028 cases) of total bacterial foodborne outbreaks (1495 cases) reported in Taiwan between 1981 and 2003 (Anon. 2005) and 31.1% of 5770 foodborne outbreaks occurred in China between 1991 and 2001 (Liu et al., 2004).
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et al., 2005). More recently, an outbreak of V. parahaemolyticus involving 177 cases occurred in the summer of 2006 was linked to contaminated oysters harvested in Washington and British Columbia (CDC, 2006). The occurrence of these outbreaks indicates that contamination of V. parahaemolyticus in oysters is a safety concern in the US.
3.2. Incidence in Europe In contrast to Asian countries, V. parahaemolyticus infections are rarely reported in European countries. However, sporadic outbreaks have been reported in countries such as Spain and France. Eight cases of V. parahaemolyticus gastroenteritis related to fish and shellfish consumption were reported in Spain In 1989 (Molero et al., 1989). An outbreak of 64 illnesses associated with raw oysters consumption occurred in 1999 in Galicia, Spain (Lozano-Leo´n et al., 2003). A serious outbreak affecting 44 patients associated with consumption of shrimps imported from Asia occurred in France in 1997 (Robert-Pillot et al., 2004). A more recent outbreak involving 80 illnesses of V. parahaemolyticus infection among guests attending weddings in one restaurant was reported in Spain in July 2004 (Martinez-Urtaza et al., 2005). Epidemiological investigation associated the outbreak with consumption of boiled crab that had been processed under unhealthy conditions. 3.3. Incidence in the United States V. parahaemolyticus was first identified as an etiological agent in the US in 1971 after three outbreaks of 425 cases of gastroenteritis associated with consumption of improperly cooked crabs occurred in Maryland (Molenda et al., 1972). Since then, sporadic outbreaks of V. parahaemolyticus infections related to consumption of raw shellfish or cooked seafood were reported throughout the US coastal regions. Between 1973 and 1998, approximately 40 outbreaks of V. parahaemolyticus infections were reported to the Centers for Disease Control and Prevention (CDC) (Daniels et al., 2000a). Among them, four major outbreaks involving more than 700 cases of illness associated with raw oyster consumption occurred in the Gulf Coast, Pacific Northwest, and Atlantic Northeast regions between 1997 and 1998. In the summer of 1997, 209 cases (including one death) of V. parahaemolyticus infections associated with raw oyster consumption occurred in the Pacific Northwest (Oregon, Washington, California and British Columbia of Canada) (CDC, 1998). In 1998, two outbreaks occurred in Washington (43 cases) and Texas (416 cases) were associated with consumption of raw oyster (DePaola et al., 2000). In addition, a small outbreak of eight cases of V. parahaemolyticus infections was reported in Connecticut, New Jersey, and New York between July and September in 1998 as a result of eating oysters and clams harvested at Long Island Sound of New York (CDC, 1999). Recently, 14 passengers on board a cruise ship in Alaska developed gastroenteritis after eating raw oysters produced in Alaska in the summer of 2004 (McLaughlin
4. Virulence factors It is known that most strains of V. parahaemolyticus isolated from the environment or seafood are not pathogenic (Nichibuchi and Kaper, 1995; FDA, 2005). Clinical strains of V. parahaemolyticus are differentiated from environmental strains by their ability to produce a thermostable direct hemolysin (TDH), an enzyme that can lyse red blood cells on Wagatsuma blood agar plates. The hemolytic activity of TDH, named the Kanagawa phenomenon, has been reported to be commonly associated with strains isolated from humans with gastroenteritis but were rarely observed in environmental isolates (Joseph et al., 1982). Therefore, the TDH has been recognized the major virulence factor of V. parahaemolyticus (Miyamoto et al., 1969; Takeda, 1983). Despite epidemiological investigations revealed a strong tie between the Kanagawa phenomenon (KP) and the pathogenicity of V. parahaemolyticus, KP-negative strains that did not produce TDH but a TDH-related hemolysin (TRH) had been isolated from outbreak patients (Honda et al., 1987, 1988). Shirai et al. (1990) examined 215 clinical strains of V. parahaemolyticus isolated from patients with diarrhea for presence of genes encoding TDH (tdh) and TRH (trh) and found that 52 strains (24.3%) carried only the trh gene. These results indicate that TRH is also a virulence factor of V. parahaemolyticus. The genes encoding TDH (tdh) and TRH (trh) have been cloned and sequenced (Kaper et al., 1984; Nishibuchi and Kaper, 1985; Taniguchi et al., 1985). Oligonucleotide probes for both tdh and trh genes have been developed for detection of virulent strains of V. parahaemolyticus (Kaper et al., 1984; Nishibuchi et al., 1986). While the pathogenicity of V. parahaemolyticus appears to be linked to the presence of tdh and trh genes, virulence factors other than TDH and TRH may yet be identified. V. parahaemolyticus strains that did not produce TDH or TRH were recently reported to induce fluid accumulation in suckling mice (Kothary et al., 2000). More recently, a heat-labile protein (serine protease) produced by a clinical V. parahaemolyticus strain carrying neither tdh nor trh gene was identified as a potential virulence factor (Lee et al., 2002). The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco-2 cells. It lysed erythrocytes and caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. However, clinical and environmental incidences of these strains have not been determined.
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6. Detection of Vibrio parahaemolyticus
In addition to the MPN method, the International Organization for Standardization (ISO) cultural method (ISO, 1990) is also widely used for detecting V. parahaemolyticus, especially in European countries. In the ISO method, test samples are incubated in two enrichment media (salt polymyxin B broth and alkaline saline peptone water or saline glucose culture medium with sodium dodecyl sulfate) at 35 1C for 7–8 h (18 h for deep-frozen products). Enriched samples are then plated on two selective media [TCBS and triphenyltetrazolium chloride soya tryptone agar (TSAT)] and incubated at 35–37 1C for 18 h (TCBS) and 20–24 h (TSAT). Colonies that are smooth, green and 2–3 mm in diameter on TCBS or are smooth, flat, dark red and 2–3 mm in diameter on TSAT are considered characteristic colonies and need be confirmed by biochemical tests. The incubation should be extended to 24 h for TCBS and 48 h for TSAT if a slow development of colonies on the media occurs. To facilitate identification of suspect isolates, several commercial biochemical test kits such as apis 20E and apis NE from BioMe´rieux (Durham, NC, USA), RapIDTM NF PLUS System from Remel Inc. (Lenexa, KS, USA), and Crystal Enteric/Non-Fermenter ID Kit (Crystal E/NF) from Becton Dickinson (Franklin Lakes, NJ, USA) are available for rapid identification of members of the family Enterobacteriaceae. These kits are fairly easy to use and can identify V. parahaemolyticus in 4 h (apis NE and RapIDTM NF PLUS), 18 h (Crystal E/NF), and 1–2 days (apis 20E). In addition, a number of automated microbial identification systems such as VITEK 2 microbial ID/AST system (BioMe´rieux) and OmniLog (Biolog, Hayward CA, USA) or semi-automated system such as MicroStation (Biolog) have been developed for identifying members of the family Enterobacteriaceae. These fully or semi-automated systems provide improved laboratory workflow efficiency and reporting accuracy. However, the initial investment of the instruments may range from $30,000 to $150,000 depending on the capacity and accessories to be included in a system.
6.1. Most probable number
6.2. Polymerase chain reaction (PCR)
The most probable number (MPN) method described in the US Food and Drug Administration Bacterial Analytical Manual (FDA, 1998) is commonly used for the detection of V. parahaemolyticus in foods. However, the MPN method is labor-intensive and time-consuming. A major disadvantage of the method is that thiosulfate– citrate–bile salts–sucrose agar (TCBS) cannot differentiate V. parahaemolyticus from some strains of Vibrio vulnificus or Vibrio mimicus. Growth of V. parahaemolyticus as well as a few strains of V. vulnificus and V. mimicus that do not ferment sucrose all appears on TCBS as round (2–3 mm in diameter) and green or blue-green colonies. Therefore, several presumptive positive colonies formed on a TCBS plate need to be analyzed with lengthy biochemical tests for confirmation and results may not be available for 4–5 days.
To overcome the disadvantage of MPN method for detecting V. parahaemolyticus, polymerase chain reaction (PCR) using DNA primers targeting tdh and trh genes encoding thermostable direct hemolysin (TDH) and TDHrelated hemolysin (TRH), respectively, were developed for detecting virulent strains of V. parahaemolyticus. Tada et al. (1992) developed a PCR procedure for specific detection of tdh and trh and reported that the procedure was capable of detecting 400 fg of cellular DNA carrying the respective gene. However, this assay cannot be used to detect V. parahaemolyticus that does not carry tdh or trh and its sensitivity was reduced by inhibitors present in normal fecal samples. Authors suggested that either a DNA extraction or an enrichment of samples in alkaline peptone water for 4 h be conducted when analyzing fecal samples.
5. Emerging strain of Vibrio parahaemolyticus Most outbreaks of V. parahaemolyticus infections were caused by V. parahaemolyticus of diverse serotypes. However, increased incidences of gastroenteritis caused by V. parahaemolyticus serotype O3:K6 have been reported in many countries since 1996 (Chiou et al., 2000; Vuddhakul et al., 2000; Gonza´lez-Escalona et al., 2005; Martinez-Urtaza et al., 2005). This serovar was first identified during a hospital-based active surveillance study of V. parahaemolyticus infections in Calcutta, India between 1994 and 1996 (Okuda et al., 1997). The survey identified a sudden increase in incidences associated with this serovar, which accounted for 63% of total V. parahaemolyticus strains isolated from patients in Calcutta between September 1996 and April 1997. This highly virulence strain was subsequently recovered at a high rate in other Southeast Asian countries and was isolated from travelers arriving in Japan from various countries in the Southeast Asia (Chiou et al., 2000; Okuda et al., 1997; Vuddhakul et al., 2000). V. parahaemolyticus O3:K6 was first identified in the US in 1998 and caused the largest outbreak (416 person) associated with oyster consumption in the US history (Daniels et al., 2000b). The same serovar was later involved in another outbreak related to shellfish consumption in Connecticut, New Jersey, and New York (CDC, 1999). Since then, a pandemic spread of this clone to other continents has been reported. In 2004, V. parahaemolyticus O3:K6 was isolates from victims of outbreaks occurred in Chile (Gonza´lez-Escalona et al., 2005) and Spain (Martinez-Urtaza et al., 2005). The isolation of the O3:K6 strain from US outbreaks raised concern about increased risks of V. parahaemolyticus infections from US shellfish consumption. However, this serovar has not been linked to illness resulted from consuming raw oysters in the US since 1999.
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The identification of a unique gene (tlh) encoding a thermolabile hemolysin (TLH) in V. parahaemolyticus (Taniguchi et al., 1985) allowed development of a multiplex PCR procedure for simultaneous detection of total and virulent V. parahaemolyticus. Although, the TLH is not considered a virulence factor of V. parahaemolyticus, the gene (tlh) encoding TLH was reported a reliable marker for V. parahaemolyticus species (Taniguchi et al., 1986). Bej et al. (1999) reported a multiplex PCR procedure for amplification of tlh, tdh and trh, which could be used for detecting total and virulent V. parahaemolyticus in shellfish. This multiplex PCR detected the tlh gene in all 111 strains of V. parahaemolyticus isolated from clinical, seafood, environmental, and oyster plants with a sensitivity for detecting all three genes of at least 1–10 cells per gram of alkaline peptone water enriched (8 h) sample homogenate. In addition to the tlh gene, other species-specific genetic markers have also been reported for detecting V. parahaemolyticus. Lee et al. (1995) cloned a 0.76-kb HindIII DNA fragment of the chromosomal DNA of V. parahaemolyticus strain 93 and reported that the cloned DNA fragment, designated pR72H, could be used as a speciesspecific DNA probe for detecting V. parahaemolyticus. The PCR assay developed for detecting the pR72H fragment was capable of detecting 2.6 fg of purified Vibrio DNA (corresponded approximately one cell) amplified through 35 cycles using primers VP33 and VP32 derived from the pR72 H fragment. Venkateswaran et al. (1998) developed a PCR procedure for detecting V. parahaemolyticus using specially designed PCR primers that amplified a 285-bp fragment within the gyrB gene (gene encoding the B subunit of DNA gyrase) of V. parahaemolyticus. Tests of 117 strains of V. parahaemolyticus isolated from various environments, food, and clinical sources with the PCR assay showed that all the strains contained the 285-bp fragment. This PCR assay was reported capable of detecting five viable V. parahaemolyticus cells (corresponding 4 pg of purified DNA). Kim et al. (1999) studied the toxR gene that involved in the regulation of many genes in Vibrio species and developed a toxR-targeted PCR for specific detection of V. parahaemolyticus. Examination of 373 strains of V. parahaemolyticus and 290 strains of other species using the PCR showed that all 373 strains of V. parahaemolyticus carried the toxR gene and gave specific amplicons, whereas no positive results were reported for non-V. parahaemolyticus in the assay. Although the PCR methods have been developed for specific detection of V. parahaemolyticus, they are limited to qualitative determination of the organism unless they are used in conjunction with the MPN procedure. Wang and Levin (2004) developed a PCR procedure for quantitative determination of total V. parahaemolyticus by analyzing the fluorescent intensity of the PCR products in the agarose gel. By comparing the fluorescent intensity of the target DNA (tlh) against the numbers of V. parahaemolyticus cells in samples, the authors reported
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a linear relationship between the fluorescent intensity of the DNA and the bacterial populations in log-value. Recent advancement in the PCR technology has lead to the development of real-time PCR for rapid and quantitative determination of V. parahaemolyticus in food samples. Kaufman et al. (2004) developed a quantitative real-time PCR assay targeting the tlh gene for enumeration of total V. parahaemolyticus and reported that the procedure could be used to quantify V. parahaemolyticus in oyster tissues and mantle fluid within 1 h of sampling time. Takahashi et al. (2005) also reported a real-time PCR method targeting the toxR gene of V. parahaemolyticus for quantitative determination of total V. parahaemolyticus in seawater and shellfish with a detection limit of 36 cells/ml in pure culture, seawater, and the homogenate of shortneck clam. 6.3. DNA hybridization In addition to PCR assays, DNA–DNA hybridization methods were also developed for specific detection of V. parahaemolyticus. Nishibuchi et al. (1985, 1986) constructed oligodeoxyribonucleotide probes labeled with P32 for detecting tdh gene in V. parahaemolyticus and found that colony hybridization test with the gene probes was more suitable than immunological assays for definitive detection of V. parahaemolyticus that produced positive reactions in the Kanagawa Phenomenon test. Lee et al. (1992) also reported a procedure using synthesized oligonucleotide probe labeled with P32 for direct detection of tdh gene of V. parahaemolyticus in artificially contaminated foods. In addition, Yamamoto et al. (1992) reported an enzyme-labeled oligonucleotide for detecting both tdh and trh genes by hybridization. Recently, two non-radioactive probes [alkaline phosphatase (AP)-labeled and digoxigenin (DIG)-labeled probes] were developed for specific and sensitive detection of tlh gene of V. parahaemolyticus (McCarthy et al., 1999). Based on the AP- and DIG-labeled probe methods, two directplating procedures using AP-and DIG-labeled probes to detect the tlh gene were subsequently developed for detecting total V. parahaemolyticus (Gooch et al., 2001). These direct-plating procedures were reported to be equivalent to a modified MPN method using AP-labeled NDA probe for detecting V. parahaemolyticus and could be completed in 1–2 days. However, it may require skilled workers to conduct the tests because both procedures involve colony lift, hybridization, and colorimetric detection with an additional preparation of probe and membrane if the digoxigenin-labeled probe method is used. More recently, Banerjee et al. (2002) developed a rapid DNA probe method for detecting V. parahaemolyticus grown on hydrophobic grid membrane filters (HGMF). In the HGMF procedure, V. parahaemolyticus is detected through hybridization between V. parahaemolyticus DNA immobilized onto HGMF and DIG-labeled probes specific for the tlh gene. Although, the method was reported
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capable of detecting V. parahaemolyticus in 1 day after an enrichment process, it is a complicate detecting system involving DNA isolation, synthesis of DIG-labeled probes, preparation of V. parahaemolyticus primers, and colony hybridization.
step procedure for quick screening of V. parahaemolyticus in foods. 7. Controls and prevention 7.1. Guidelines
6.4. Chromogenic medium Although PCR and DNA probe methods are available for specific detection of total or virulent V. parahaemolyticus, most of them require special instruments and skilled technicians to conduct the assays. A simple procedure using traditional plating technique for detecting V. parahaemolyticus needs to be developed to allow persons with limited microbiological training to conduct routine screening of the organism in foods or environmental samples. Recently, a chromogenic medium (Bio-Chrome Vibrio medium) was developed to allow differentiation of V. parahaemolyticus from other Vibrio species based on formation of unique purple colonies on the medium. Growth of V. parahaemolyticus on Bio-Chrome Vibrio medium (BCVM) can easily be distinguished from bluegreen colonies formed by growth of V. vulnificus, V. cholerae and V. mimicus. A study of evaluating selectivity and specificity of BCVM using 179 strains of Vibrio spp. found that BCVM was capable of differentiating V. parahaemolyticus from other species including V. vulnificus and V. mimicus. All 148 strains of V. parahaemolyticus grew on BCVM and 145 of them produced purple colonies. The remaining 31 Vibrio spp., except one strain of V. fluvialis, were either unable to grow, or produced bluegreen or white colonies on BCVM (Su et al., 2005). Duan and Su (2005b) compared BCVM with TCBS for detecting V. parahaemolyticus in 296 seawater, sediment, and oyster samples using a 3-tube MPN method and found that the specificities of BCVM and TCBS for V. parahaemolyticus detection were 94% and 77%, respectively, while the accuracies of both media for detecting V. parahaemolyticus were 84% (BCVM) and 54% (TCBS). These results indicate that BCVM is more specific and accurate than TCBS for detecting V. parahaemolyticus. Subsequently, a double layer agar plate (DLAP), based on the thin agar layer (TAL) method (Kang and Fung, 2000), was developed as a one-step procedure for direct enumeration of V. parahaemolyticus (Duan et al., 2006). The DLAP, prepared by overlaying an equal volume (10 ml) of a non-selective medium (tryptic soy agar supplemented with 1.5% NaCl) on top of the selective BCVM, was capable of detecting V. parahaemolyticus in mixed cultures containing non-Vibrio bacteria based on the formation of purple colonies. Direct plating on DLAP was found as effective as the MPN method for recovering heat- and coldinjured V. parahaemolyticus cells, which could not be detected by direct plating on BCVM or TCBS. The DLAP offers an alternative to MPN method for detecting injured V. parahaemolyticus cells and can be used as a simple, one-
The ubiquitous nature of Vibrio species in marine and estuarine environments makes it impossible to obtain seafood free of theses bacteria. Following the outbreaks of V. parahaemolyticus infection occurred in 1997 and 1998, the US Food and Drug Administration (FDA) provided guidance and recommendations to Interstate Shellfish Sanitation Conference (ISSC) for monitoring V. parahaemolyticus, which limit viable V. parahaemolyticus to 10,000 or fewer cells per gram of seafood (ISSC, 1997). However, examination of shellfish samples by state and federal authorities following the 1998 outbreaks found that overall levels of V. parahaemolyticus in most oysters from implicated growing areas were less than 1000 Vibrio cells per gram with some of them as low as 100 cells per gram (Kaysner and DePaola, 2000). These findings suggest a possible low infectious dose of the pathogenic strains involved in the outbreaks and that the FDA guidance may not be sufficient to protect consumers from V. parahaemolyticus infection associated with raw oyster consumption. To limit growth of V. parahaemolyticus in contaminated oysters, the National Shellfish Sanitation Program Guide for the Control of Molluscan Shellfish established time-totemperature regulations that limit the maximum time of exposure of oysters to elevated temperatures. Shellfish harvested for raw consumption need to be cooled down to 10 1C (50 1F) within 10, 12, and 36 h of harvest when the average monthly maximum air temperature is X27 1C (81 1F), between 19 and 27 1C (66–80 1F), and o18 1C (66 1F), respectively (NSSP, 2003). In an attempt to reduce V. parahaemolyticus infection associated with oyster consumption, harvest of oysters in Mississippi for raw consumption is limited from mid-September to April. Oyster beds are closed during the warmer summer months and at other times when heavy rains cause an influx of potentially contaminated water (Andrews, 2004). These harvesting practices minimized the incidence of V. parahaemolyticus illness from consumption of Mississippi oysters. In addition to temperature controls, harvest practices can also influence levels of V. parahaemolyticus in oysters harvested with the intertidal practice. In the intertidal harvest, oysters are exposed to ambient air, which allows V. parahaemolyticus to multiply rapidly in oysters especially on warm and sunny days. Nordstrom et al. (2004) investigated effect of low-tide exposure of oysters to ambient conditions on V. parahaemolyticus levels in oysters and found that the mean densities of V. parahaemolyticus in oysters were four to eight times greater after maximum exposure than at the corresponding initial exposure. The
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study demonstrated that the densities of V. parahaemolyticus could increase in oysters during the low-tide exposure in summer and suggested a modification of the harvest practice, such as avoiding harvest of oysters after intertidal exposure to ambient conditions, could potentially reduce the incidence of V. parahaemolyticus infection associated with raw oyster consumption.
7.2. Relaying and depuration Relaying and depuration are common approaches for reducing bacterial contaminants in shellfish. In the relaying process, shellfish is transferred before harvest from polluted areas to an unpolluted waterway for natural biological purification. However, increased pollution along coastal line due to introduction of animal waste from farmland into marine environment has resulted in reduced clean area for growing shellfish. The lack of clean and unpolluted marine environment for growing shellfish creates a big challenge for the relaying practice. Depuration is a controlled process that allows shellfish to purge sand and grit from the gut into clean seawater. The process usually leads to a reduction of microbial contaminants in shellfish and therefore increases shelf life of refrigerated products. However, studies have shown that depuration with clean seawater was not effective in reducing certain persistent bacteria including Vibrio spp. in shellfish because of the colonization of those bacteria in the intestinal tracts. In most instances, total aerobic plate counts could be reduced by one log value via the depuration process. Nevertheless, reduction of bacteria to fewer than 104 cells per gram of shellfish is rarely reported. A study conducted by Kelly and Dinuzzo (1985) reported that oysters required 16 days to depurate laboratorycontaminated V. vulnificus to non-detectable level. In a similar study, Eyles and Davey (1984) observed no significant differences in mean counts of naturally occurring V. parahaemolyticus between depurated and nondepurated oysters. To increase the efficacy in reducing bacterial contamination in oysters, depuration in conjunction with chlorine, ultraviolet light, ozone or iodophors were studied (Fleet, 1978). However, none of them could effectively eliminate V. parahaemolyticus from shellfish. Croci et al. (2002) studied depuration of blue mussels experimentally contaminated with Escherichia coli, Vibrio cholerae and V. parahaemolyticus in ozonated water and reported a substantially smaller reduction in the numbers of V. cholerae and V. parahaemolyticus in the mussels (approximately 1 log) than of E. coli (approximately 3 log) after 44 h of process. Ren and Su (2006) examined the effects of electrolyzed oxidizing (EO) water depuration on reducing V. parahaemolyticus and V. vulnificus in laboratory-contaminated oysters and found that both species could only be reduced by approximately 1.0-log unit after 8 h at room temperature.
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7.3. Thermal processes Thermal processes such as cold storage, freezing, and low temperature pasteurization have been reported capable of achieving certain reductions of Vibrio species in oysters. Thompson and Vanderzant (1976) reported that populations of V. parahaemolyticus in shucked oysters decreased from 411,000 to 0.36 MPN/g after 7 days of storage at 3 1C. Muntada-Garriga et al. (1995) reported that viable cells of V. parahaemolyticus (105–7 cfu/g) in oyster homogenates were completely inactivated by freezing at 18 and 24 1C for 15–28 weeks depending on initial populations of the microorganism and freezing temperatures. Andrews et al. (2000) developed a lowtemperature pasteurization for shellstock oysters by placing the oysters in 55 1C water to achieve an internal temperature of 48–50 1C for 5 min. The authors reported that the process reduced V. parahaemolyticus in oysters (1.2 105 MPN/g) to non-detectable levels (o3 MPN/g). An added benefit of the mild heat treatment is that oysters are often killed and shucked automatically during the treatment. Therefore, oysters need to be banded before being processed to prevent loss of juice during treatments. A major disadvantage of the pasteurization process is that it may cause changes in oyster texture due to protein denaturation occurred during the heat treatment. 7.4. High-pressure processing High hydrostatic pressure is a non-thermal process that can be used to destroy pathogenic microorganisms in foods and extend shelf life of food products without apparent changes in original nutrients, flavor, and appearance. Studies have shown that high-pressure processing (HPP) was effective in inactivating V. parahaemolyticus in oysters. Cook (2003) reported that a HPP treatment of 300 MPa for 180 s was sufficient to achieve a 45-log reduction of V. parahaemolyticus, including V. parahaemolyticus O3:K6 strains, in oysters. Calik et al. (2002) found that treatments of 345 MPa for 30 and 90 s were optimum conditions for reducing V. parahaemolyticus in pure culture (7.6 106–5.5 108 cfu/ml) and in oysters (8.4 105–3.4 107 cfu/g), respectively, to nondetectable levels (o10 cfu/mL or cfu/g). Similar to the mild heat treatment, HPP treatment also assists in oyster shucking by destroying the adduct muscle. Oysters also need to be banded to prevent opening of shell during the treatment. He et al. (2002) reported that HPP treatments of 240–275 MPa were optimum for shucking Pacific oyster with minimum changes in appearance. The main disadvantage of applying HPP for reducing V. parahaemolyticus in oysters is the high costs of initial investment of the high-pressure system, which is not easily affordable by most oyster producers. 7.5. Irradiation Irradiation is another non-thermal process capable of destroying bacterial pathogens in foods. Studies have
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shown that low dosages (o3 kGy) of irradiation did not kill oysters or affect the sensory quality of oysters (Jakabi et al., 2003). Andrews et al. (2003) reported that naturally incurred V. vulnificus (3-log/g) in oysters was reduced to non-detectable levels with a Cobalt-60 gamma radiation treatment at 0.75 kGy. Treatments of oysters inoculated with V. parahaemolyticus O3:K6 (104 cell/g) with 1.0–1.5 kGy also reduced the contamination to nondetectable levels. The investigators concluded that oysters treated with o2 kGy had a high oyster survival rate and did not significantly affect the sensory quality. Sensory analysis conducted with 146 volunteers using difference tests was unable to distinguish irradiated oysters from nonirradiated ones. Although irradiation appears to be an effective means for eliminating V. parahaemolyticus in oysters, the reluctance among consumers to accept food irradiated with radioactive sources and the need to safely handle radioactive materials limit its usage. 8. Conclusion Food-borne illness caused by V. parahaemolyticus, particularly O3:K6 strain, has increased globally over the last 10 years. Even though two hemolysins (TDH and TRH) have been well recognized as virulence factors for V. parahaemolyticus, a comprehensive understanding of the mechanism that the organism infects human remains to be determined. A recent statement of Quantitative Risk Assessment on the Public Health Impact of Pathogenic V. parahaemolyticus in Raw Oysters released by FDA (2005) identified deficiencies of current knowledge in incidence/frequency of pathogenic V. parahaemolyticus in water and shellfish, impact of overnight submersion of intertidally harvested oysters, growth rate of V. parahaemolyticus, impact of post-harvest handling and processing, and impact of consumer handling of raw oysters. Future research needs to be conducted to gather scientific data to answer these uncertainties. Acknowledgments Preparation of this article is supported by the National Oceanic and Atmospheric Administration (NOAA) Grant NA130A and by Shanghai Leading Academic Discipline Project T1102. References Alam, M.J., Tomochika, K.I., Miyoshi, S.I., Shinoda, S., 2002. Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan. FEMS Microbiol. Lett. 208, 83–87. Andrews, L.S., 2004. Strategies to control Vibrios in molluscan shellfish. Food Prot. Trends 24, 70–76. Andrews, L.S., Park, D.L., Chen, Y.P., 2000. Low temperature pasteurization to reduce the risk of Vibrio infections from raw shellstock oysters. Food Addit. Contam. 17, 787–791.
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