Interleukin 2 Secretion by T Cells for Detection of Biologically Active Staphylococcal Enterotoxin Type E.

Staphylococcus aureus is a significant worldwide source of clinical infections and foodborne illnesses; it acts through the synthesis of a group of enterotoxins (SEs) that cause gastroenteritis and also function as superantigens that activate T cells, resulting in massive cytokine production, yielding life-threatening toxicity. It is important that methods for detection and quantification of these toxins respond to their activity and not just the presence of the toxin molecule, which may be deactivated. Traditionally, live animals have been used to test for emesis following administration of the toxin-containing sample. Here, we present results studying cell-based alternatives for the assay of active staphylococcal enterotoxin type E (SEE), a toxin subtype identified in foodborne outbreaks in the United States, the United Kingdom, and France. We found that interleukin 2 production by T cells can be used as a specific biological marker for the quantitative detection of SEE as compared with subtypes SEA and SEB. Our assay shows a dose-response relationship between IL-2 secretion by Jurkat T-cell line and SEE concentration as low as 1 pg/mL.

Rapid Cell-Based Assay for Detection and Quantification of Active Staphylococcal Enterotoxin Type D.

Food poisoning by Staphylococcus aureus is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by this bacterium and is a major source of foodborne illness. Staphylococcal enterotoxin D (SED) is one of the predominant enterotoxins recovered in Staphylococcal food poisoning incidences, including a recent outbreak in Guam affecting 300 children. Current immunology methods for SED detection cannot distinguish between the biologically active form of the toxin, which poses a threat, from the inactive form, which poses no threat. In vivo bioassays that measure emetic activity in kitten and monkeys have been used, but these methods rely upon expensive procedures using live animals and raising ethical concerns. A rapid (5 h) quantitative bioluminescence assay, using a genetically engineered T-cell Jurkat cell line expressing luciferase under regulation of nuclear factor of activated T cells response elements, in combination with the lymphoblastoid B-cell line Raji for antigen presentation, was developed. In this assay, the detection limit of biologically active SED is 100 ng/mL, which is 10 times more sensitive than the splenocyte proliferation assay, and 105 times more sensitive than monkey or kitten bioassay. Pasteurization or repeated freeze-thaw cycles had no effect on SED activity, but reduction in SED activity was shown with heat treatment at 100°C for 5 min. It was also shown that milk exhibits a protective effect on SED. This bioluminescence assay may also be used to rapidly evaluate antibodies to SED for potential therapeutic application as a measurement of neutralizing biological effects of SED.

Low cost quantitative digital imaging as an alternative to qualitative in vivo bioassays for analysis of active aflatoxin B1.

Aflatoxin B1 (AFB1) producing fungi contaminate food and feed and are a major health concern. To minimize the sources and incidence of AFB1 illness there is a need to develop affordable, sensitive mobile devices for detection of active AFB1. In the present study we used a low cost fluorescence detector and describe two quantitative assays for detection of detoxified and active AFB1 demonstrating that AFB1 concentration can be measured as intensity of fluorescence. When the assay plate containing increasing concentrations of AFB1 is illuminated with a 366 nm ultraviolet lamp, AFB1 molecules absorb photons and emit blue light with peak wavelength of 432 nm. The fluorescence intensity increased in dose dependent manner. However, this method cannot distinguish between active AFB1 which poses a threat to health, and the detoxified AFB1 which exhibits no toxicity. To measure the toxin activity, we used a cell based assay that makes quantification more robust and is capable of detecting multiple samples simultaneously. It is an alternative to the qualitative duckling bioassay which is the "gold-standard" assay currently being used for quantitative analysis of active AFB1. AFB1 was incubated with transduced Vero cells expressing the green fluorescence protein (GFP) gene. After excitation with blue light at 475 nm, cells emitted green light with emission peak at 509 nm. The result shows that AFB1 inhibits protein expression in a concentration dependent manner resulting in proportionately less GFP fluorescence in cells exposed to AFB1. The result also indicates strong positive linear relationship with R(2)=0.90 between the low cost CCD camera and a fluorometer, which costs 100 times more than a CCD camera. This new analytical method for measuring active AFB1 is low in cost and combined with in vitro assay, is quantitative. It also does not require the use of animals and may be useful especially for laboratories in regions with limited resources.

New Stx2e Monoclonal Antibodies for Immunological Detection and Distinction of Stx2 Subtypes.

BACKGROUND: Stx2e is a primary virulence factor in STEC strains that cause edema disease in neonatal piglets. Though Stx2a and Stx2e are similar, many antibody-based Stx detection kits are designed to detect Stx2a and do not recognize the Stx2e subtype. METHODS AND FINDINGS: Four monoclonal antibodies against Stx2e were developed and characterized. Two of these mAbs recognize the B subunit of Stx2e, Stx2f, and to a lesser extent, Stx2b, Stx2c, and Stx2d. The other two mAbs recognize the A subunit of Stx2e, and cross-react with all Stx2 subtypes except Stx2f. The most sensitive sandwich ELISA using these mAbs has a limit of detection for Stx2e of 11.8 pg/mL. The ability of the neutralizing antibody Stx2e-2 to block Stx2e-receptor binding in Vero cells was visualized using immunofluorescence. Combinations of these and previously developed mAbs permit ELISA-based differentiation between closely related Stx2a, Stx2c, and Stx2d (using mAbs Stx2-5/2-1, Stx2-5/2e-2, and Stx2e-3/2e-2, respectively). CONCLUSIONS: The sensitive immunoassays developed in this study should augment our capacity to detect Stx2e in porcine environments and biological samples. Moreover, immunoassays that can distinguish between the closely related Stx2a, Stx2c, and Stx2d subtypes can be useful in quickly analyzing Stx subtypes in samples containing more than one strain of STEC.

Intracellular trehalose and sorbitol synergistically promoting cell viability of a biocontrol yeast, Pichia anomala, for aflatoxin reduction.

Pichia anomala (Wickerhamomyces anomalus) WRL-076 was discovered by a visual screening bioassay for its antagonism against Aspergillus flavus. The yeast was shown to significantly inhibit aflatoxin production and the growth of A. flavus. P. anomala is a potential biocontrol agent for reduction of aflatoxin in the food chain. Maintaining the viability of biocontrol agents in formulated products is a great challenge for commercial applications. Four media, NYG, NYGS, NYGT and NYGST are described which support good growth of yeast cells and were tested as storage formulations. Post growth supplement of 5 % trehalose to NYGST resulted in 83 % viable yeast cells after 12 months in cold storage. Intracellular sorbitol and trehalose concentrations were determined by HPLC analysis at the beginning of the storage and at the end of 12 month. Correlation of cell viability to both trehalose and sorbitol suggested a synergistic effect. Bonferroni (Dunn) t Test, Tukey's Studentized Range (HSD) Test and Duncan's Multiple Range Test, all showed that yeast cell viability in samples with both intracellular trehalose and sorbitol were significantly higher than those with either or none, at a 95 % confidence level. DiBAC4(5) and CFDA-AM were used as the membrane integrity fluorescent stains to create a two-color vital staining scheme with red and green fluorescence, respectively. Yeast cells stored in formulations NYG and NYGS with no detectable trehalose, displayed mostly red fluorescence. Yeast cells in NYGST+5T showed mostly green fluorescence.

Sensitive detection of active Shiga toxin using low cost CCD based optical detector.

To reduce the sources and incidence of food-borne illness there is a need to develop affordable, sensitive devices for detection of active toxins, such as Shiga toxin type 2 (Stx2). Currently the widely used methods for measuring Shiga toxin are immunoassay that cannot distinguish between the active form of the toxin, which poses a threat to life, to the inactive form which can bind to antibodies but show no toxicity. In this work, we determine toxin activity based on Shiga toxin inhibition of green fluorescent protein (GFP) combined with low cost charge-coupled device (CCD) fluorescence detection, which is more clinically relevant than immunoassay. For assay detection, a simple low cost fluorescence detection system was constructed using a CCD camera and light emitting diode (LED) excitation source, to measure GFP expression. The system was evaluated and compared to a commercial fluorometer using photomultiplier detection for detecting active Stx2 in the range 100 ng/mL-0.01 pg/mL. The result shows that there is a negative linear relationship between Stx2 concentrations and luminous intensity of GFP, imaged by the CCD camera (R(2)=0.85) or fluorometer (R(2)=0.86). The low cost (∼$300) CCD camera is capable of detecting Shiga toxin activity at comparable levels as a more expensive (∼$30,000) fluorometer. These results demonstrate the utility and the potential of low cost detectors for toxin activity; this approach may increase the availability of foodborne bacterial toxin diagnostics in regions where there are limited resources and could be readily adapted to the detection of other food-borne toxins.

Rapid detection of predation of Escherichia coli O157:H7 and sorting of bacterivorous Tetrahymena by flow cytometry.

Protozoa are known to harbor bacterial pathogens, alter their survival in the environment and make them hypervirulent. Rapid non-culture based detection methods are required to determine the environmental survival and transport of enteric pathogens from point sources such as dairies and feedlots to food crops grown in proximity. Grazing studies were performed on a soil isolate of Tetrahymena fed green fluorescent protein (GFP) expressing Escherichia coli O157:H7 to determine the suitability of the use of such fluorescent prey bacteria to locate and sort bacterivorous protozoa by flow cytometry. In order to overcome autofluorescence of the target organism and to clearly discern Tetrahymena with ingested prey vs. those without, a ratio of prey to host of at least 100:1 was determined to be preferable. Under these conditions, we successfully sorted the two populations using short 5-45 min exposures of the prey and verified the internalization of E. coli O157:H7 cells in protozoa by confocal microscopy. This technique can be easily adopted for environmental monitoring of rates of enteric pathogen destruction vs. protection in protozoa.

Quantitative analysis of staphylococcus enterotoxin A by differential expression of IFN-γ in splenocyte and CD4⁺ T-cells.

Staphylococcus aureus is an important bacterial pathogen that produces a range of Staphylococcal Enterotoxins (SEs) which cause gastroenteritis and superantigen activation of T cells, the mechanism of which is not well understood. The ability to rapidly detect and quantify SEs is very important in order to learn the causes of staphylococcal outbreaks and to stop similar outbreaks in the future. Enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of several SEs. However, these immunological methods cannot distinguish between active and inactive toxin. It is known that interferon-gamma (IFN-γ) expressed in response to stimulation by SEs contributes significantly to the pathogenesis of S. aureus infection. Nonetheless, the cellular source of IFN-γ is still unclear and the contributions of the specific splenocyte types. In our effort to understand the immunologic response to Staphylococcal Enterotoxin A (SEA) exposure, we studied IFN-γ production in mouse splenocytes. We demonstrated that short term ex vivo exposure of splenocytes or primary naïve CD4+ T-cells to biologically active SEA induces differential expression of IFN-γ mRNA in a time and dose dependent manner and the expression levels reflect the levels of IFN-γ secreted protein. Positive isolated CD4+ T-cells accounted for only 10% of IFN-γ production. We also demonstrate that expression of IFN-γ can be used for rapid quantitative analysis of active SEA with a detection limit of 1 ng/mL.

Bacterial communities in urban aerosols collected with wetted-wall cyclonic samplers and seasonal fluctuations of live and culturable airborne bacteria.

Airborne transmission of bacterial pathogens from point sources (e.g., ranches, dairy waste treatment facilities) to areas of food production (farms) has been suspected. Determining the incidence, transport and viability of extremely low levels of pathogens require collection of high volumes of air and characterization of live bacteria from aerosols. We monitored the numbers of culturable bacteria in urban aerosols on 21 separate days during a 9 month period using high volume cyclonic samplers at an elevation of 6 m above ground level. Culturable bacteria in aerosols fluctuated from 3 CFU to 6 million CFU/L of air per hour and correlated significantly with changes in seasonal temperatures, but not with humidity or wind speed. Concentrations of viable bacteria determined by fluorescence staining and flow cytometry correlated significantly with culturable bacteria. Members of the phylum Proteobacteria constituted 98% of the bacterial community, which was characterized using 16S rRNA gene sequencing using DNA from aerosols. Aquabacterium sp., previously characterized from aquatic environments, represented 63% of all clones and the second most common were Burkholderia sp; these are ubiquitous in nature and some are potential human pathogens. Whole genome amplification prior to sequencing resulted in a substantial decrease in species diversity compared to characterizing culturable bacteria sorted by flow cytometry based on scatter signals. Although 27 isolated colonies were characterized, we were able to culture 38% of bacteria characterized by sequencing. The whole genome amplification method amplified DNA preferentially from Phyllobacterium myrsinacearum, a minor member of the bacterial communities, whereas Variovorax paradoxus dominated the cultured organisms.

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 naïve 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.

Application of flow cytometry and cell sorting to the bacterial analysis of environmental aerosol samples.

Flow cytometry (FCM) combined with viability staining is a useful tool in discerning viable bacteria in environmental samples where traditional culture methods may fail. Contamination of aerosol samples with dust and other non-biological particles can interfere with accurate sample analysis and therefore there is a desire to exclude those particles from analysis. Particles were sorted according to their light scattering properties, cultured and isolates obtained. Isolates were cultured in suspension and reanalyzed by flow cytometry. The isolates were also analyzed and identified by DNA sequence analysis. Isolates with statistically similar light scattering properties shared common sequence identification. Isolates exhibited distinct light scattering profiles that roughly correlated with their originating gate, but often the peak of the profile was outside that gate.

Electrolytic destruction of urea in dilute chloride solution using DSA electrodes in a recycled batch cell.

The destruction of urea, its fate and influence on levels of active chlorine as well as formation of byproducts during electrolytic treatment of urea solutions was studied. Treated solutions designed to simulate typical concentrations in dairy manure lagoon water, contained initial concentrations of urea up to 750 ppm (mg L(-1)), chloride from 100 to 400 ppm, and 2000 ppm NaHCO(3). Current from 0.6 to 2.5A (34--141Am(-2)) was applied using oxide-coated titanium electrodes in a bench scale electrolysis device. Current and energy efficiencies of urea destruction increased with temperature and initial concentrations of chloride and urea. These parameters also affected the concentrations of oxidized chlorine species during treatment. Residual nitrogen was comprised of a constant fraction (0.07) of urea nitrogen oxidized to nitrate over the temperature range studied (16--34.5 degrees C). Chlorate was also formed as a byproduct of electrolysis.