Edema of the paws of white mice--test for assessing the activity of E. coli enterotoxins]

The authors studied the paw edema test in mice for detection of the E. coli (strain P-99) enterotoxins activity. This test proved to be simple, sensitive and reproducible; it permitted to determine the activity of thermostable and thermolabile enterotoxins and endotoxin; the mentioned test was particularly useful in testing various preparations of enterotoxins obtained during their extraction and purification.     

Use of the toxin-tissue receptor reaction for detecting toxic substances of the causative agents of acute intestinal diseases

The possibility of using erythrocytic ganglioside diagnostic reagents (EGDR) for the detection of V. cholerae, E. coli and S. typhimurium enterotoxins in the passive hemagglutination (PHA) test has been shown. Museum strains and cultures isolated from patients with acute intestinal diseases were tested for the presence of enterotoxins. Cell-free extracts were studied by biological methods and by serological titration in the PHA test with the use of EGDR. The diagnostic reagent was found to interact only with those enterotoxins whose specific receptors were gangliosides GM1.     

Comparison of three immunological methods for detecting staphylococcal enterotoxins from food

AIMS: Immunologically based assays for the detection of staphylococcal enterotoxins are numerous. These techniques include radio immunosorbent assays and enzyme-linked immunosorbent assays (ELISA), some of which are available as commercial kits. The purpose of this study was to compare the performances of three commercial immunoassays. METHODS AND RESULTS: Two automated detection systems, VIDAS SET bioMèrieux, VIDAS SET2 bioMérieux and an ELISA method, TRANSIA PLATE Staphylococcal Enterotoxins Diffchamb were compared for detecting different quantities of purified staphylococcal enterotoxins (A, B, C2, D and E) added to food. CONCLUSIONS: VIDAS SET2 had a greater specificity (100%) and sensitivity than VIDAS SET and TRANSIA PLATE Staphylococcal Enterotoxins. More precisely, VIDAS SET2 could detect <0.5 ng g(-1) of toxins A and B, <1 ng g(-1) of toxins C2 and E and 1 ng g(-1) of toxins D and E. SIGNIFICANCE AND IMPACT OF THE STUDY: Because staphylococcal food poisoning (resulting from ingestion of low levels of staphylococcal enterotoxins) is one of the most common forms of foodborne illness there is a need for specific and sensitive methods for detecting these enterotoxins. VIDAS SET2 appears to be suitable for detecting staphylococcal enterotoxins from food.     

Ecology of spontaneous fermentation in one winery during 5 consecutive years

A concentration protocol based on trichloroacetic acid precipitation was evaluated and compared with the reference method using dialysis concentration. Different quantities of purified staphylococcal enterotoxins were added to pasteurized Camembert-type cheeses. Detection of enterotoxins in these cheeses was performed using an automated detection system. Raw goat milk Camembert-type cheeses involved in a staphylococcal food poisoning were also tested. Both enterotoxin extraction methods allowed detection of the lowest enterotoxin concentration level used in this study (0.5 ng g-1). Compared with the dialysis concentration method, TCA precipitation of staphylococcal enterotoxins was 'user-friendly' and less time-consuming. These results suggest that TCA precipitation is a rapid (1 h), simple and reliable method of extracting enterotoxin from food which gives excellent recovery from dairy products.     

Detection of staphylococcal enterotoxins A, B, C, D, and E in foods by radioimnunoassay, using staphyloccal cells containing protein A as immunoadsorbent.

A sensitive radioimmunoassay utilizing Staphylococcus aureus cells containing protein A as a coprecipitant was developed for the detection and quantitation of staphylococcal enterotoxins A, B, C, D, and E in a variety of foods. The enterotoxins were extracted from the foods by a simple and rapid procedure. The sensitivity of the assay is 1.0 ng or less of enterotoxin per g of food.     

Novel Platform for the Detection of Staphylococcus aureus Enterotoxin B in Foods

Staphylococcal contamination of food products and staphylococcal food-borne illnesses continue to be a problem worldwide. Screening of food for the presence of Staphylococcus aureus and/or enterotoxins using traditional methods is laborious. Reliable and rapid multiplex detection methods from a single food extract or culture supernatant would simplify testing. A fluorescence-based cytometric bead array was developed for the detection of staphylococcal enterotoxin B (SEB), using magnetic microspheres coupled with either an engineered, enterotoxin-specific Vβ domain of the T-cell receptor (Vβ-TCR) or polyclonal antibodies. The binding affinity of the Vβ-TCR for SEB has been shown to be in the picomolar range, comparable to the best monoclonal antibodies. The coupled beads were validated with purified enterotoxins and tested in a variety of food matrices spiked with enterotoxins. The Vβ-TCR or antibody was shown to specifically bind SEB in four different food matrices, including milk, mashed potatoes, vanilla pudding, and cooked chicken. The use of traditional polyclonal antibodies and Vβ-TCR provides a redundant system that ensures accurate identification of the enterotoxin, and the use of labeled microspheres permits simultaneous testing of multiple enterotoxins from a single sample.     

Protein Standard Absolute Quantification (PSAQ) for improved investigation of staphylococcal food poisoning outbreaks

Staphylococcal enterotoxins are major causing agents of food-borne diseases. Their detection in food remnants for risk assessment or food poisoning outbreaks investigation suffers from a lack in comprehensive immunological tools. In this study, we demonstrate that the combination of immunocapture and Protein Standard Absolute Quantification (PSAQ) strategy, which uses isotope-labeled enterotoxins as internal standards for MS-based analysis, is powerful to specifically identify and quantify these contaminating agents in food matrices. This approach is believed to significantly improve the elucidation of staphylococcal food poisoning outbreaks.     

Evaluation of the reversed passive latex agglutination (RPLA) test kits for detection of staphylococcal enterotoxins A, B, C, and D in foods

Four lots of the SET-RPLA kit (Denka Seiken Co. Ltd., Tokyo), a commercial reverse passive latex agglutination test kit for the detection of staphylococcal enterotoxins A, B, C, and D in foods, have been evaluated for their efficacy. The kits showed high specificity and sensitivity with a detection limit of 0.75 ng enterotoxin/g of food. The test is simple, is completed within 24 h, and does not require complicated extraction or concentration procedures nor expensive equipment. In addition, the assay is semiquantitative. However, as in any other immunological system, routine verification of the specificity of the latex reagents against standard enterotoxins and toxin-free food extracts is necessary.     

Detection of enterotoxin and toxic shock syndrome toxin 1 genes in Staphylococcus, with emphasis on coagulase-negative staphylococci

The detection of staphylococcal enterotoxins is decisive for the confirmation of an outbreak and for the determination of the enterotoxigenicity of strains. Since the recognition of their antigenicity, a large number of serological methods for the detection of enterotoxins in food and culture media have been proposed. Since immunological methods require detectable amounts of toxin, molecular biology techniques represent important tools in the microbiology laboratory. In the present study, polymerase chain reaction (PCR) was used to identify genes responsible for the production of enterotoxins and toxic shock syndrome toxin 1 (TSST-1) in S. aureus and coagulase-negative staphylococci (CNS) isolated from patients and the results were compared with those obtained by the reverse passive latex agglutination (RPLA) assay. PCR detection of toxin genes revealed a higher percentage of toxigenic S. aureus strains (46.7%) than the RPLA method (38.3%). Analysis of the toxigenic profile of CNS strains showed that 26.7% of the isolates produced some type of toxin, and one or more toxin-specific genes were detected in 40% of the isolates. These results suggests the need for further studies in order to better characterize the pathogenic potential of CNS and indicate that attention should be paid to the toxigenic capacity of this group of microorganisms.     

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.     

Enterotoxins of Staphylococcus aureus induce a cytopathic effect in cell lines.

We describe the use of cell cultures for the detection of Staphylococcus aureus enterotoxins. Madin Darby Bovine Kidney (MDBK), Bovine Embryo lung cells (PEB) and Dog Carcinoma Cell line (A-72) cell monolayers were tested. MDBK, A-72 and PEB cell lines proved to be susceptible to the cytotoxic effect induced by staphylococcal enterotoxins. A cytopathic effect was observed in some cases after 2 hours of incubation. The PEB cell line was the most susceptible and may be used as a useful and cheap method for screening enterotoxigenic S. aureus strains.     

CD154 as a potential early molecular biomarker for rapid quantification analysis of active Staphylococcus enterotoxin A

Staphylococcus aureus is a major bacterial pathogen producing a group of 21 enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis, and they function as superantigens that activate large numbers of T cells. In the current study, we demonstrate that short-term ex vivo exposure of primary na?ve CD4(+) T-cells to SEA induces differential expression of the T cell surface receptor CD154 in a time- and dose-dependent manner. In addition, we show that SEA induces higher CD154 protein expression and higher splenocyte cell proliferation compared with SEB. We also demonstrate that expression of CD154 can be used for rapid detection of active SEA in milk.     

A Novel ELISA Format for the Rapid and Sensitive Detection of Staphylococcal Enterotoxin A

Staphylococcal food poisoning is one of the leading causes of bacterial food poisoning each year. Detection kits for staphylococcal enterotoxins are commercially available and the assays can require from one and a half to twenty-four hours to complete with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food. We have successfully demonstrated a microsphere-packed capillary (MPC) ELISA for the detection of staphylococcal enterotoxin A (SEA) and have compared it to two commercially available kits. The MPC assay detected a lower amount of SEA in ham, chicken, cheese, and bean sprouts than either of the two commercially available kits. In addition, the novel MPC assay was completed in less than ten minutes, as compared to three and twenty-four hours for the two commercially available kits. This research also demonstrated that the MPC ELISA can contain integrated positive and negative controls and has the potential to simultaneously detect and identify multiple enterotoxins.     

Rapid detection of staphylococcal enterotoxins in foods with a modification of the reversed passive latex agglutination assay

A modification of the reversed passive latex agglutination kit assay for detection of staphylococcal enterotoxins by centrifugation of microtitration plates reduces the incubation time of the assay from 20-24 h to 4 h. Enterotoxins can therefore be detected in foods within the working day.     

Rapid detection of staphylococcal enterotoxins in foods with a modification of the reversed passive latex agglutination assay

A modification of the reversed passive latex agglutination kit assay for detection of staphylococcal enterotoxins by centrifugation of microtitration plates reduces the incubation time of the assay from 20–24 h to 4 h. Enterotoxins can therefore be detected in foods within the working day.     

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.     

Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.     

Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.     

Comparison of different purification procedure for extraction of staphylococcal enterotoxin A from foods.

Different procedures commonly used for extraction, purification, and concentration of staphylococcal enterotoxins from foods were investigated with 131I- and 125I-labeled staphylococcal enterotoxin A. Loss of labeled enterotoxin A was compared with loss of total nitrogen. The results showed that in most of the common procedures, such as gel filtration, ion exchange, and heat treatment, the percentage of loss of labeled enterotoxin A was greater than the loss of total nitrogen. Chloroform extraction and acid precipitation with hydrochloric acid had nearly the same effect on the purification of both labeled enterotoxin A and total nitrogen. Ammonium sulfate precipitation proved to be practical and was successfully used for purification of enterotoxin A from sausage extract. Simultaneous use of trypsin and Pseudomonas peptidase for treatment of food extracts considerably reduced food proteins capable of interfering with serological detection of enterotoxins but did not essentailly influence the loss of enterotoxin A.     

Capillary Tube Immunological Assay for Staphylococcal Enterotoxins

A simple assay is reported in which 1 mug of staphylococcal enterotoxins A, B, and D per ml was detected in less than 1 hr. Interfacial reaction of antisera and enterotoxin solutions in a 1-ml internal diameter capillary tube allowed rapid detection of sera type.