Results and Discussion

SFP is caused by ingestion of food containing SE preformed by metabolically active staphylococci. SFP is usually a self-limiting illness, often abrupt and severe in onset, with a short incubation period (1 to 8 h).

Victims experience severe nausea and characteristi-cally projectile vomiting (Balaban and Rasooly, 2000;

Dinges et al., 2000; Le Loir et al., 2003). The severity depends on an individual’s susceptibility to the SE, the amount of contaminated food eaten, the amount of toxin in the food ingested, and the general health of the victim. Other symptoms include convulsive retch-ing, diarrhea, abdominal cramps, pain, headache, and muscle ache. The illness may be accompanied by sub-normal temperature and transient changes in blood emetic (Bergdoll, 1988). The cysteine loop is not

directly associated with emesis and is believed to pro-vide proper orientation of critical residues located near the disulfi de linkage (Warren et al., 1974). SEA, SEC1, SEC2, SEC3, SED, SEE, and SElH bind zinc which is required by some toxins to bind major histo-compatibility complex class II (MHC II) (Baker and Acharya, 2004) (see below). Depending on the toxin, zinc binding can occur at the outer surface or in a classical motif (H-E-X-X-H) in the groove between two domains (Baker and Acharya, 2004).

SAg Activity

SEs and SEls belong to a family of toxins pro-duced by S. aureus and Streptococcus pyogenes, which share several properties, including superanti-gen (SAg) activity. SAgs stimulate a high percentage of T cells. Most bind to the outside of the peptide binding groove of MHC II molecules on antigen-pre-senting cells (APCs) and to T-cell receptors (TCRs) bearing specifi c beta chain V sequences on T cells.

Binding activates APCs and extensive proliferation of T cells, resulting in an acute uncontrolled release of proinfl ammatory cytokines and immunomodulatory cytokines in a chronic response (McCormick et al., 2001). Recent studies demonstrated that long-term stimulation by SAgs induces development of CD4^ CD25^ regulatory T cells in humans, rodents, and bovines, capable of suppressing the responses of responder T-cell proliferation (Seo et al., 2007). This suggests that immunomodulation induced by SAgs has an important role in staphylococcal diseases.

Emesis

Emesis involves coordinated gastrointestinal activity, including contraction of abdominal muscles, forceful contractions of the stomach pylorus, and relaxation of the fundus, cardiac sphincter, and esophagus. Studies with primates demonstrated that SEs stimulate neural receptors in the abdomen, which transmit impulses through the vagus and sympathetic nerves, ultimately stimulating the central vomiting center in the fourth ventricle (Sugiyama and Hayama, 1965). However, little is known about the molecular and cellular mechanisms by which SEs induce emesis.

This gap in knowledge is due to a lack of a convenient and inexpensive animal model confi rmed to mimic human SFP following oral administration. The mon-key feeding assay described by Bergdoll (1988) is con-sidered to be the standard method. Other animal models, including kittens, piglets, house musk shrews, and ferrets, have been used (Hu et al., 2007). Results with these models, particularly those that require sys-temic administration rather than oral feeding, must

2000). There has been one report of a U.S. outbreak by shredded pork and coleslaw contaminated with a community-acquired methicillin-resistant S. aureus, which is known to frequently express SEs (Jones et al., 2002). Due to the lack of a unifi ed surveillance system for the European Union, the reported preva-lence of SFP varies greatly from country to country.

SFP accounted for an average of 4.5% of food-borne disease cases (926 outbreaks) between 1993 and 1998 in 15 European Union countries (WHO Surveil-lance Programme, 2000). In Japan, 2,525 outbreaks were reported, involving 59,964 persons between 1980 and 1999 (Shimizu et al., 2000).

Distribution of SEs among Staphylococci Many SE and SEl genes are associated with genomic islands and mobile genetic elements such as the staphylococcal pathogenicity island (designated SaPI), prophages, or plasmids (Baba et al., 2002).

The known locations of SE and SEl genes are sum-marized in Table 2. Recent studies demonstrated that SaPI is mobilized by staphylococcal bacteriophages and endogenous prophages (Lindsay et al., 1998), suggesting that SE genes might be transferred hori-zontally. Numerous studies have assessed SE and SEl profi les of S. aureus from various sources. Although there is considerable variability among strains from different geographic regions, the most commonly observed SEs are those harbored on the enterotoxin gene cluster (egc), namely, those encoded by the sei, sem, sen, and seo genes with or without the seg gene, followed by those on the genetic element named bovine staphylococcal pathogenicity island (SaPI bov) (sec, tst, and sel) (Fitzgerald et al., 2000; Kwon et al., 2004; Lawrynowicz-Paciorek et al., 2007; Srinivasan et al., 2006). In contrast, the SE most frequently pro-duced by S. aureus implicated in SFP is SEA, followed by SED, SEI, and SEC (Chiang et al., 2008; Kerouan-ton et al., 2007). Another coagulase-positive species, S. intermedius, is a well-known SE producer that pos-sesses a variant SEC gene, seccanine, as well as genes for pressure and pulse rate. Typically, the duration of SFP

varies from several hours to 1 day. Dehydration, pros-tration, and shock can occur in severe cases which may require hospitalization. Deaths are rare, with fatality rates ranging from 0.03% for the general pub-lic to 4.4% for the very young, the elderly, or chroni-cally ill patients. Fatal cases are usually associated with fl uid and metabolic abnormalities, despite intra-venous electrolyte and fl uid replacement. It is esti-mated that ~1,750 hospitalizations and two deaths typically occur each year from SFP in the United States (Mead et al., 1999).

SFP Incidence

SFP occurs as isolated cases or outbreaks.

Although it has likely affl icted humankind for centu-ries, the exact incidence is unknown for several rea-sons. Although there is a national surveillance system for food-borne disease in the United States through FoodNet (CDC, 1998), SFP is not offi cially report-able. In addition, since it is not usually life-threaten-ing, defi nitive diagnoses of isolated cases are not usually accomplished, or affected individuals may not seek treatment. Confi rmatory diagnosis is not always practical since it usually requires identifi ca-tion of SE-producing S. aureus in food which may have been discarded. Since SFP is not an infection, attempts to isolate the causative staphylococci from patients are not warranted.

Outbreaks typically receive considerable public-ity. Some examples of recent outbreaks are summa-rized in Table 3. In the United States, 47 outbreaks of SFP involving 3,181 cases were reported between 1983 and 1987 and 42 reported outbreaks involving 1,413 cases occurred between 1993 and 1997, accounting for 1.6% of total food-borne disease cases (Olsen et al., 2000). It has been estimated that the mean annual number of cases from 1982 through 1997 could have been 185,060 (Mead et al., 1999), and the cost of SFP in the United States is approxi-mately $1.5 billion annually (Harvey and Gilmour,

Table 3. Large outbreaks of SFP

Yr Site or country Product No. of cases Reference

1975 Commercial airline Omelets and ham for airplane meal in Anchorage, AL

197 State of Alaska (1975)

1983 Caribbean cruise ship Cream-fi lled pastries 215 CDC (1983) 1986 United States Low-fat chocolate milk ca. 1,000 Evenson et al. (1988)

1990 Thailand Éclairs 485 Thaikruea et al. (1995)

1992 United States Chicken salad 1,364 USFDA (1992)

2000 Japan Low-fat milk 14,870 Asao et al. (2003)

2004 Brazil Ordination meal ca. 8,000 Do Carmo et al. (2004)

of SE genes using degenerate primers, followed by characterization of the amplicons by microchip hybridization with oligonucleotide probes specifi c for SE genes. This assay has the advantage that it can detect previously unidentifi ed SE genes.

Despite the usefulness of methods described above, their performance time and compromised accuracy due to food matrices limit their effective-ness. Recently the ability of biosensors to overcome these shortcomings has been investigated. These sys-tems typically link a biological component, such as an antibody, with a physicochemical transducer to convert and amplify minute signals from the biologi-cal component into a measurable signal. Enzymatic bio-nanotransduction systems use specifi c antibody conjugated to nano-signal-producing DNA templates.

Signals may be amplifi ed by in vitro transcription of DNA templates bound to target molecules producing RNA nano-signals specifi c for multiple targets in the sample. Recent application of this method detected SEB at a level of 0.11 ng/ml (Branen et al., 2007).

Biomolecular interaction analysis mass spectrometry was developed and applied to detect SEB and TSST-1.

Biomolecular interaction analysis-mass spectometry uses surface plasmon resonance to detect SEB binding to antibody immobilized on a gold electrode, fol-lowed by matrix-assisted laser desorption ionization–

time-of-fl ight to identify and differentiate the bound toxins. This method detected and differentiated SEB and TSST-1 at levels of 1 ng/ml in milk and mush-rooms (Nedelkov et al., 2000).

CONCLUDING REMARKS

SFP has been a major concern of the food indus-try and medical profession for many decades, and great advances have been made in regard to its pre-vention, treatment, diagnosis, and etiology. Although it is not usually life-threatening, SFP produces consid-erable morbidity and economic impact. An under-standing of the exact impact in the United States would require more stringent and uniform reporting requirements. In recent years, major advances were made in understanding the complicated epidemiology of SFP, and several past dogmas are now considered obsolete. For example, we have only recently learned that, in addition to the fi ve classical SEs, many addi-tional molecular variants and recently identifi ed SEs and SEls exist. Some of these related proteins are con-fi rmed to lack emetic activity, suggesting that emesis is not necessarily a feature of the toxins that provides a selective advantage to staphylococci. It is more likely that enterotoxicity is a secondary effect of SE-induced immunomodulation and activity as SAgs.

other SEs (SEA, SEB, SEC, SED, and SEE) and a novel SE-related gene, se-int (Futagawa-Saito et al., 2004). A 1991 outbreak of SFP in the United States involving 265 persons was conclusively linked to an SEA-producing strain of S. intermedius (Khambaty et al., 1994). Therefore, staphylococci other than S. aureus should not be neglected in regard to risk of SFP.

In fact, CNS also harbor SE genes, albeit less fre-quently than S. aureus. S. xylosus and S. cohnii from dry cured ham produce SEC and SED (Rodriguez, 1996). S. equorum, S. capitis, S. lentus, S. gallinarum, and S. cohnii from goat milk and cheese produce SEE (Vernozy-Rozand et al., 1996). S. capitis, S. cohnii, S. haemolyticus, S. hominis, S. hyicus subspecies hyi-cus, S. saprophytihyi-cus, S. schleiferi, S. warneri, and S. xylosus from various sources reportedly produce SEs (Bautista et al., 1988; Breckinridge and Bergdoll, 1971; Hoover et al., 1983; Olsvik et al., 1982; Udo et al., 1999; Valle et al., 1990). It is unclear whether these species have the capacity to frequently cause SFP since one study reported that CNS produce very small amounts of SE (10 ng/ml) (Bergdoll, 1995).

SE Detection Methods

There is considerable interest in detecting SEs or their genes for epidemiological purposes. Cur-rently, commercial kits are available to detect the classical SEs (SEA to SEE) in food. These are usually based on passive agglutination or the enzyme-linked immunosorbent assay and include the SET-RPLA kit (Denka Seiken), the visual immunoassay (Tecra), BioDetect Test Strip (Alexeter Technologies), Tran-siaTube (Diffchamb AB), and Ridascreen (rBio-pharma). Detection of more recently identifi ed SEs is problematic, since fewer reagents are available, par-ticularly antisera. T-cell mitogenicity has been used to detect novel SEs based on their SAg activity (Holt-freter et al., 2004). Some laboratories have raised antibodies for the detection of certain SEs, such as SEG, SElH, and SEI (Omoe et al., 2002).

PCR is an extremely powerful tool to rapidly screen S. aureus strains for SE-encoding genes. Sev-eral multiplex PCR methods have been developed.

Monday and Bohach (1999) developed an assay which, in a single reaction, detects nine SE genes.

Others extended this method and developed PCR techniques for larger numbers of SE genes in multiple reactions (Omoe et al., 2005b). Multiplex PCR meth-ods that subtype the sec gene into its variants sec1, sec2, and sec3 have been described (Chen et al., 2001). Letertre et al. (2003b) developed a real-time PCR to detect the sea to sej genes. Sergeev et al. (2004) described an assay which involves PCR amplifi cation

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Presumably, both biological properties are endowed by the unique and conserved molecular structures of SEs, which have been revealed in the past decade. The identifi cation of novel toxins and improved methods of detection have also indicated that SE production is much more widespread than originally thought and includes species of staphylococci other than S. aureus.

One key area of understanding that is still lacking includes the molecular and cellular mechanisms by which SEs induce SFP. It is becoming clear that induc-tion of emesis and SAg activity are not directly related, but the lack of a convenient and inexpensive animal model has hindered progress in this area.

Acknowledgments. Our efforts in preparation of the manuscript were supported by grants from the PHS (U54AI57141, RR15587, and RR00166) and the Idaho Agricultural Experiment Station.

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