Development of an in vitro activity assay as an alternative to the mouse bioassay for Clostridium botulinum neurotoxin type A.

Currently, the only accepted assay with which to detect active Clostridium botulinum neurotoxin is an in vivo mouse bioassay. The mouse bioassay is sensitive and robust and does not require specialized equipment. However, the mouse bioassay is slow and not practical in many settings, and it results in the death of animals. Here, we describe an in vitro cleavage assay for SNAP-25 (synaptosome-associated proteins of 25 kDa) for measuring the toxin activity with the same sensitivity as that of the mouse bioassay. Moreover, this assay is far more rapid, can be automated and adapted to many laboratory settings, and has the potential to be used for toxin typing. The assay has two main steps. The first step consists of immunoseparation and concentration of the toxin, using immunomagnetic beads with monoclonal antibodies directed against the 100-kDa heavy chain subunit, and the second step consists of a cleavage assay targeting the SNAP-25 peptide of the toxin, labeled with fluorescent dyes and detected as a fluorescence resonance energy transfer assay. Our results suggest that the sensitivity of this assay is 10 pg/ml, which is similar to the sensitivity of the mouse bioassay, and this test can detect the activity of the toxin in carrot juice and beef. These results suggest that the assay has a potential use as an alternative to the mouse bioassay for analysis of C. botulinum type A neurotoxin.

Detection of botulinum neurotoxin-A activity in food by peptide cleavage assay.

The World Health Organization (WHO) and U.S. Centers for Disease Control and Prevention (CDC) have labeled botulinum toxins as a high priority biological agent that may be used in terrorist attacks against food supplies. Due to this threat there is an increased need to develop fast and effective methods to detect active botulinum neurotoxins (BoNTs). This study reports the successful use of an enzymatic assay employing an internally quenched fluorogenic peptide as a fast, simple and inexpensive alternative to the mouse bioassay. In less than 15 min the assay can detect 0.25 nM BoNT-A in liquid food samples. The detection level is far below the adult human lethal oral dose of 70 microg of toxin. Immunomagnetic beads coated with IgG monoclonal antibodies that target the toxin heavy chain can concentrate the toxin without neutralizing its enzymatic activity, overcoming matrix effects caused by endogenous protease inhibitors and peptidases. This fast and effective assay system could be used for large scale screening to detect BoNT-A.

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.

The olive compound 4-hydroxytyrosol inactivates Staphylococcus aureus bacteria and Staphylococcal Enterotoxin A (SEA).

The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single chain protein which consists of 233 amino acid residues with a molecular weight of 27078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, diarrhea, atopic dermatitis, arthritis, and toxic shock) syndromes. Changes in the native structural integrity may inactivate the toxin by preventing molecular interaction with cell membrane receptor sites of their host cells. In the present study, we evaluated the ability of the pure olive compound 4-hydroxytyrosol and a commercial olive powder called Hidrox-12, prepared by freeze-drying olive juice, to inhibit S. aureus bacteria and SEA's biological activity. Dilutions of both test substances inactivated the pathogens. Two independent cell assays (BrdU incorporation into newly synthesized DNA and glycyl-phenylalanyl-aminofluorocoumarin proteolysis) demonstrated that the olive compound 4-hydroxytyrosol also inactivated the biological activity of SEA at concentrations that were not toxic to the spleen cells. However, efforts to determine inhibition of the toxin by Hidrox-12 were not successful because the olive powder was cytotoxic to the spleen cells at concentrations found to be effective against the bacteria. The results suggest that food-compatible and safe antitoxin olive compounds can be used to inactivate both pathogens and toxins produced by the pathogens. Practical Application: The results of this study suggest that food-compatible and safe antitoxin olive compounds can be used to reduce both pathogens and toxins produced by the pathogens in foods.

Clostridium botulinum neurotoxin type B is heat-stable in milk and not inactivated by pasteurization.

Foodborne botulism is caused by the ingestion of foods containing botulinum neurotoxins (BoNTs). To study the heat stability of Clostridium botulinum neurotoxins, we needed to measure and compare the activity of botulinum neurotoxins, serotypes A and B, under various pasteurization conditions. Currently, the only accepted assay to detect active C. botulinum neurotoxin is an in vivo mouse bioassay, which raises ethical concerns with regard to the use of experimental animals. In this study, noninvasive methods were used to simultaneously detect and distinguish between active BoNT serotypes A and B in one reaction and sample. We developed an enzymatic activity assay employing internally quenched fluorogenic peptides corresponding to SNAP-25, for BoNT-A, and VAMP2, for BoNT-B, as an alternative method to the mouse bioassay. Because each peptide is labeled with different fluorophores, we were able to distinguish between these two toxins. We used this method to analyze the heat stability of BoNT-A and BoNT-B. This study reports that conventional milk pasteurization (63 °C, 30 min) inactivated BoNT serotype A; however, serotype B is heat-stable in milk and not inactivated by pasteurization. Using this activity assay, we also showed that the commonly used food processes such as acidity and pasteurization, which are known to inhibit C. botulinum growth and toxin production, are more effective in inactivating BoNT serotype A than serotype B when conventional pasteurization (63 °C, 30 min) is used.

Shiga toxin Stx2 is heat-stable and not inactivated by pasteurization.

Shiga toxin-producing Escherichia coli have been associated with food-borne illnesses. Pasteurization is used to inhibit microbial growth in milk, and an open question is whether milk pasteurization inactivates Shiga toxins. To answer this question we measured Shiga toxin's inhibition effect on Vero cell dehydrogenase activity and protein synthesis. Our data demonstrate that Shiga toxin 2 (Stx2) is heat-stable and that pasteurization of milk, at the various suggested temperatures and times by the U.S. Food and Drug Administration, (63 degrees C for 30 min, or 72 degrees C for 15s or 89 degrees C for 1s), did not reduce the biological activity of Stx2. However, treatment at 100 degrees C for 5 min inactivated the toxin. These data demonstrate that Stx2 is not inactivated by conventional pasteurization.

Inhibition of biological activity of staphylococcal enterotoxin A (SEA) by apple juice and apple polyphenols.

The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single-chain protein that consists of 233 amino acid residues with a molecular weight of 27 078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, diarrhea, atopic dermatitis, arthritis, and toxic shock) syndromes. Changes of the native structural integrity may inactivate the toxin by preventing molecular interaction with cell membrane receptor sites of their host cells. In the present study, we evaluated the ability of one commercial and two freshly prepared apple juices and a commercial apple polyphenol preparation (Apple Poly) to inhibit the biological activity of SEA. Dilutions of freshly prepared apple juices and Apple Poly inhibited the biological activity of SEA without any significant cytotoxic effect on the spleen cells. Additional studies with antibody-coated immunomagnetic beads bearing specific antibodies against the toxin revealed that SEA added to apple juice appears to be largely irreversibly bound to the juice constituents. The results suggest that food-compatible and safe anti-toxin phenolic compounds can be used to inactivate SEA in vitro and possibly also in vivo, even after induction of T-cell proliferation by long-term exposure to SEA. The significance of the results for microbial food safety and human health is discussed.

Inhibition of Shiga toxin 2 (Stx2) in apple juices and its resistance to pasteurization.

In the present study, we evaluated Shiga toxin (Stx2) activity in apple juices by measuring a decrease in dehydrogenase activity of Vero cells with the microculture tetrazolium (MTT) assay. Freshly prepared juice from Red Delicious apples and Golden Delicious apples inhibited the biological activity of the bacterial toxin Stx2 produced by E. coli O157:H7 strains. Studies with immunomagnetic beads bearing specific antibodies against the toxin revealed that Stx2 activity was restored when removed from the apple juice. SDS gel electrophoresis revealed no difference (P < 0.05) in the densities or molecular weights between Stx2 in either PBS or apple juices. These results suggest that Stx2 may be reversibly bound to small molecular weight constituents in the juice. The Stx2 toxin was not inactivated on exposure to heat programs (63 degrees C for 30 min, 72 degrees C for 15 s, 89 degrees C for 1 s) commonly used to pasteurize apple juice, but lost all activity when exposed to 100 degrees C for 5 min. The results suggest that pasteurization of apple juice used to inactivate E. coli O157:H7 has no effect on Stx2, and that food-compatible and safe antitoxin compounds can be used to inhibit the biological activity of the Shiga toxin.

Ingested Shiga toxin 2 (Stx2) causes histopathological changes in kidney, spleen, and thymus tissues and mortality in mice.

The Shiga toxin (Stx)-producing bacterial strain, Escherichia coli O157:H7, colonizes the distal small intestine and the colon, initiating serious illness, including hemolytic-uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Although intravenous administration of purified Stx to primates has been able to reproduce the features of HUS, it has not been conclusively established as to whether ingestion of Stx alone without the bacterium poses a potential health risk. To help answer this question, in this study, we fed Shiga toxin 2 (Stx2) directly into the stomachs of mice via gavage. Our data show that ingestion of Stx2 at a concentration of 50 µg/mouse induces weight loss and kills the mice at 3-5 days post-gavage. Additional studies revealed that the toxin retains activity at low pH, that its activity is neutralized by treatment with toxin-specific antibody, and that about 1% of the fed toxin is absorbed into the blood circulation. Lethality by intraperitoneal (IP) injection of Stx2 occurred at much lower doses than by ingestion. Detailed histopathological evaluation of stained tissues by light microscopy revealed severe histopathological changes in kidneys, spleen, and thymus but not in the pancreas, lymph nodes, heart, lungs, trachea, esophagus, stomach, duodenum, jejunum, ileum, cecum, and colon. The pathological changes in the kidney appeared similar to those seen in humans with HUS. The cited data suggest that (a) most but not all of the toxin is inactivated in the digestive tract, (b) part of the oral-ingested toxin is absorbed from the digestive tract into the circulation, (c) enough active toxin reaches susceptible organs to induce damage, and (d) Stx2 in the absence of toxin-producing bacteria can be harmful to mice. The results are clinically relevant for food safety because we also found that heat treatments (pasteurization) that destroy bacteria did not inactivate the heat-resistant toxin produced and secreted by the bacteria.

In vitro cell-based assay for activity analysis of staphylococcal enterotoxin A in food.

Staphylococcal enterotoxins (SEs) are a leading cause of food poisoning and have two separate biological activities; it causes gastroenteritis and functions as a superantigen that activates large numbers of T cells. In vivo monkey or kitten bioassays were developed for analysis of SEs emetic activity. To overcome the inherent limitations of such bioassays, this study describes an in vitro splenocyte proliferation assay based on SEs superantigen activity as an alternative method for measuring the activity of staphylococcal enterotoxin A (SEA). After incubation of splenocytes with SEA, cell proliferation was measured by labeling the proliferating cells' DNA with bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) and quantifying the incorporated BrdU by immunohistochemistry. BrdU labeling is shown to be highly correlated with SEA concentration (R(2)=0.99) and can detect 20 pg mL(-1) of SEA, which is far more sensitive than most enzyme-linked immunosorbent assays. Our assay can also distinguish between active toxin and inactive forms of the toxin in milk. By applying immunomagnetic beads that capture and concentrate the toxin, our assay was able to overcome matrix interference. These results suggest that our in vitro cell-based assay is an advantageous practical alternative to the in vivo monkey or kitten bioassays for measuring SEA and possibly other SEs activity in food.