Raising Antibodies to Staphylococcal Enterotoxins A and B

A method is described for raising specific antibodies to staphylococcal enterotoxins A (SEA) and B (SEB) by intravenous inoculation of rabbits with small doses of enterotoxin diluted in sterile physiological saline. A course of six injections over a period of two weeks was given on four occasions. After the third course, 3/5 rabbits given SEA had a titre ± 1/50 and 4/6 given SEB had a titre of 1/50. Titres were not appreciably enhanced by the fourth series of injections. Only minor non-specific reactions which would require no absorption were found at the end of the series. It is believed that the findings in this study would be reproducible and that the method is likely to be suitable for raising antisera to other enterotoxins. Reference is also made to preliminary experiments in which latex sensitized with SEA or SEB was inoculated intravenously into rabbits.     

Staphylococcal Enterotoxins B and C

Abstract

The staphylococcal enterotoxins (SEs) are a subgroup of related protein exotoxins in the pyrogenic toxin (PT) family produced by Staphylococcus aureus and Streptococcus pyogenes (1). Like other members of the PT family, the SEs are superantigens and elaborate a set of biological activities linked to their ability to stimulate cells of the immune system (2). These activities contribute to their ability to induce toxic shock syndrome, immunosuppression, and probably other diseases (3). However, as is evident from the fact that they are designated as enterotoxins, the SEs are distinguishable from other members of the PT family by their ability to induce gastroenteritis when ingested. Hence, they are the causative agents in staphylococcal food poisoning (SFP), a very common form of food-associated gastroenteritis in the United States and worldwide (4).     

Staphylococcal Enterotoxins A and B: Solid-Phase Radioimmunoassay in Food

An immunoassay employing (125)I labeled enterotoxins A and B and polystyrene tubes coated with specific antibodies was used for detection and quantitation of enterotoxin in food. Ham salad, cheddar cheese, custard, condensed milk, and salami were studied. Enterotoxin was successfully determined in all the foods by simple extraction procedures. The assay was sensitive to 1 to 10 ng of toxin per g of food; nonspecific inhibitions were 15% or less.     

Thermal Inactivation of Staphylococcal Enterotoxins B and C

Thermal inactivation profiles of staphylococcal enterotoxins B (SEB) and C (SEC) at 80, 100, and 121 C showed that SEC is more resistant than SEB to heat. After 24 h of incubation at 25 C, some reactivation (recovery of serological reactivity) occurred in toxins that had been inactivated by heat. If the toxin was stirred during heating, reactivation did not occur. An examination of the reactivation kinetics of heat-treated SEC showed that reactivation was temperature dependent. At 25 C, the incubation temperature of heat-treated crude SEC (80 C for 10 min), 100% reactivation occurred after 24 h, whereas at 4 C only slight reactivation was observed. We and others observed that heat-treated toxins initially lost more serological activity when heated at a low temperature (80 C) than at a higher temperature (100 C); in the present study we demonstrate that this is a reversible phenomenon.     

Capillary Tube Assay for Staphylococcal Enterotoxins A, B, and C

A miniaturized single-gel diffusion procedure for detection of staphylococcal enterotoxin is proposed. The technique effects substantial savings of reagents and is easy to perform.     

Production of Staphylococcal Enterotoxins A, B, and C in Various Media

The effect of initial pH and of length of incubation time at 37 C in four different growth media on the production of staphylococcal enterotoxins A, B, and C was determined. A starting pH of 6.8 gave higher yields of enterotoxins B and C than either pH 6.0 or 5.3. The production of enterotoxin A was, however, not materially affected by the low initial pH of 5.3. Prolonged incubation (48 to 72 hr) resulted only occasionally in higher yields of enterotoxin. The effect of the media on the amount of enterotoxin produced is considerable. Difco Brain Heart Infusion (BHI) was inferior to either Fisher BHI, 4% NZ Amine (NAK), or 3% NAK plus 3% protein hydrolysate powder at the three initial pH values, regardless of length of incubation time. The slight effect of the low starting pH on the production of enterotoxin A is being further investigated.     

Production of Staphylococcal Enterotoxins A, B, and C in Colloidal Dispersions

Larger amounts of enterotoxin were produced when Staphylococcus aureus S-6 was grown under still (nonshaken) conditions in a medium that was a paste or gel than were produced in a liquid dispersion with the same colloidal ingredient or in control basal broth (4% NZ Amine-NAK containing 50 mug of thiamine per 100 ml and 1 mg of niacin per 100 ml). Four colloidal ingredients were used which had been previously demonstrated to not support enterotoxin production in buffer. The effect of the type of dispersion occurred earlier than that of the colloidal ingredient, but interactions were found. This effect was not observed when the cells were grown with aeration (shaken). Four other strains of S. aureus followed a similar pattern for enterotoxins A, B, and C, although liquid and paste with cornstarch and carrageenan were the only media compared to the control broth. Enterotoxins A and B were produced earlier by S. aureus S-6, and much greater quantities of enterotoxins were produced for all strains when incubated shaken.     

Staphylococcal Enterotoxins A,D, and E

Abstract

Staphylococcal enterotoxins (SEs) are a family of structurally related exotoxin molecules produced by certain Gram-positive Staphylococcus aureus bacterial strains. SEs are a major cause of food poisoning and are involved in bacterial Gram-positive shock in humans. SEs bind to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) and subsequently activate a large fraction, 5–20%, of T lymphocytes (1). This property has led to their classification of superantigens (SAg). The T cells are activated by SAg to proliferate and produce cytokines such as interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor-α and β (TNF-α and β) (2,3). Depending on origin, superantigens can be divided in two groups, viral and bacterial. For reviews see Refs. 4–8.     

金黄色葡萄球菌肠毒素A、B、C、D、E重组载体的构建及表达

金黄色葡萄球菌肠毒素（Staphylococcal enterotoxins,SEs）是一组结构毒力相似、血清型不同的可溶性小分子蛋白质,平均分子质量为26~30 kD,是引起细菌性食物中毒及肠胃炎的主要因素之一。为了制备金黄色葡萄球菌肠毒素的纯品,首先合成了SEA、SEB、SEC、SED和SEE的基因序列,然后构建了5种肠毒素的原核表达载体,分别转入BL21（DE3）细胞中进行诱导表达。通过SDS-PAGE和Western blot验证,5种肠毒素蛋白均被成功表达,并且在较低的诱导温度（16 ℃）获得一定量的可溶性蛋白。成功制备了5种金黄色葡萄球菌肠毒素的可溶性蛋白,为今后更好地解决因SEs引起的食品安全问题奠定了基础。     

Polyvalent Antiserum Agar System for the Detection of Staphylococcal Enterotoxins A, B, C, and E

A polyvalent antiserum agar system in capillary tubes was developed and evaluated for the detection of enterotoxins A, B, C, and/or E present in culture supernatant fluids.     

Stability of 125I-Labeled Staphylococcal Enterotoxins in Solid-Phase Radioimmunoassay

Staphylococcal enterotoxins, Types A, B, and C, were labeled with 1252 by the chloramine-T method at approximately two levels of specific activity, 40 and 4 muCi/mug of protein. Toxins labeled with high specific activity showed extensive dissociation of 125I when stored at different temperatures, including -23 C. In contrast, toxins labeled with low specific activity did not show any significant loss of 125I when stored at -23 C for as long as 2 months. Enterotoxins, whether labeled with high or low activities, formed aggregates immediately upon labeling. Aggregate formation increased in high-activity-labeled toxins on storage at -23 C, and low-activity-labeled toxins showed no significant increase in aggregate formation, even after 2 months at -23 C. The aggregated forms of the enterotoxins were either devoid of antigenic activity in solid-phase radioimmunoassay or they possessed significantly reduced antigenic activity. Thus, a decrease in binding of 1252-labeled enterotoxin to specific antibody in solid-phase radioimmunoassay results mainly from (i) loss of 125I on storage, and (ii) formation of aggregates with reduced antigenic activity.     

Mass Outbreak of Food Poisoning Disease Caused by Small Amounts of Staphylococcal Enterotoxins A and H

It was believed that food poisoning in Osaka in 2000 was due to small amounts of staphylococcal enterotoxin A (SEA) in reconstituted milk. Results of this study clearly indicate that SEH was also present in the raw material of reconstituted milk, indicating that the food poisoning was caused by multiple staphylococcal enterotoxins.     

Improved Reversed Passive Hemagglutination for Simple and Rapid Detection of Staphylococcal Enterotoxins A~E in Food

Detection and identification of staphylococcal enterotoxins in food or culture filtrates were performed using the reversed passive hemagglutination (RPHA) technique, with formalized sheep red blood cells (FSRBC) sensitized with immunoglobulins of anti-A, B, C, D, and E rabbit hyperimmune sera fractionated by affinity chromatography. The FSRBC sensitized with anti-A~E immunoglobulins showed a high level of reactivity and specificity in RPHA, against homologous types of purified enterotoxins and culture filtrates of toxin-producing strains. No non-specific reactions with various ingredients in foods nor cross-reactions among enterotoxin types were observed. The minimum amount of enterotoxins in foods detected by RPHA was calculated to be 0.01 μg/g without concentration, and the recovery rate of experimentally added toxins was calculated to be about 80%. Under routine laboratory practice, detection and identification of enterotoxins from incriminated foods of five food poisoning outbreaks were performed by RPHA within 3 hr after reception of the specimens. Among them, three were determined to be enterotoxin A food poisoning, one to be toxin C and the rest to be intoxication of A and D. The concentration of the toxins was between 0.014 and 3.65 μg per gram of food.     

Innovative Application of Mass Spectrometry for the Characterization of Staphylococcal Enterotoxins Involved in Food Poisoning Outbreaks

Staphylococcal poisoning is a common food-borne disease for which immunoassays to detect enterotoxins were developed, but these assays often lead to false diagnoses due to interferences or lack of specificity. Absolute quantitative mass spectrometry was for the first time successfully applied to an investigation of a staphylococcal outbreak due to coconut pearls.     

豚鼠和BALB/c小鼠对SEC2超抗原作用敏感性的比较

比较分析豚鼠和BALB/c小鼠对金黄色葡萄球菌肠毒素C2(Staphylococcus enterotoxin C2,SEC2)超抗原作用的敏感性差异,确定更适合用于SEC2超抗原作用研究的模式动物。体外试验,以梯度浓度SEC2刺激豚鼠和BALB/c小鼠的外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)和脾淋巴细胞,MTS染色法检测其增殖;体内实验,SEC2腹腔注射豚鼠和BALB/c小鼠,每隔3 d检测豚鼠和小鼠的体重、体温变化情况;每隔7 d采血,ELISA法检测血清中IL-4、IFN-γ、IL-2和TNF-α分泌水平。不同浓度的SEC2均能刺激小鼠的PBMC和脾淋巴细胞显著增殖(P0.05),而对豚鼠的PBMC和脾淋巴细胞无效。腹腔注射后,SEC2组和对照组豚鼠和小鼠的体温均无显著性变化(P0.05);豚鼠SEC2组体重在给药后期显著低于对照组(P0.05),小鼠的SEC2组体重在给药后第3-30天均显著低于对照组(P0.05),此后恢复到正常水平(P0.05);给药后各时间点,小鼠的SEC2组血清中细胞因子IL-4、IFN-γ、IL-2和TNF-α的分泌水平均显著高于对照组(P0.05),而豚鼠的SEC2组血清中细胞因子分泌水平与对照组无显著性差异(P0.05)。与豚鼠相比,BALB/c小鼠对SEC2超抗原活性的敏感程度更高,且表现出很好的剂量和时间效应,适合作为模式动物用于SEC2的超抗原活性研究。     

Biosensor Detection of Botulinum Toxoid A and Staphylococcal Enterotoxin B in Food

Immunoassays were developed for the simultaneous detection of staphylococcal enterotoxin B and botulinum toxoid A in buffer, with limits of detection of 0.1 ng/ml and 20 ng/ml, respectively. The toxins were also spiked and measured in a variety of food samples, including canned tomatoes, sweet corn, green beans, mushrooms, and tuna.     

Staphylococcal enterotoxin B induces anergy to conventional peptide in memory T cells

Microbial superantigens can alter host immunity through aberrant activation and subsequent anergy of responding naive T cells. We show here that the superantigen, staphylococcal enterotoxin B (SEB), directly induces tolerance in memory CD4 T cells. Murine naive and memory CD4(+) T cells were labeled with the fluorescent dye CFSE and the cells were exposed to SEB before they were cultured with specific peptide antigen. Memory, but not naive, T cells became anergic and did not respond to their cognate peptide antigen. The extent and duration of T cell receptor (TCR) clustering was similar to promote naive T cell activation and memory T cell anergy, suggesting similar TCR-SEB interactions led to distinct intracellular signaling processes in the two cell types. Like SEB, soluble anti-CD3 mAb does not stimulate memory cell proliferation. However, unlike SEB, soluble anti-CD3 mAbs did not induce anergy to cognate peptide. Anergy was directly visualized in vivo. CD4(+) memory T cells were identified in mice that had been administered SEB. The cells failed to proliferate in response to subsequent immunization with their cognate recall antigen. Hence, one mode of pathogen survival is the modulation of host immunity through selective elimination of memory T cell responses.