Egg yolk antibodies for detection and neutralization of Clostridium botulinum type A neurotoxin.

The objective of this research project was to determine the usefulness of an egg antibody platform for producing materials for the detection and neutralization of botulinum type A neurotoxin. Yield estimates for detection and neutralizing antibodies produced using methods described were calculated. Antibody specific to botulinum toxoid A (aToxoid) and toxin A (aBoNT/A) was produced by immunizing hens with botulinum toxoid A (toxoid) followed by increasing amounts of botulinum neurotoxin A (BoNT/A) in Freund incomplete adjuvant. Egg yolks were extracted with polyethylene glycol (PEG) for antibody detection and neutralization experiments. A model aToxoid/toxoid immunoassay using only egg yolk antibody was developed and had a detection limit of 1 pg/ml of toxoid. In an indirect enzyme-linked immunosorbent assay of BoNT/A-specific antibody, the aBoNT/A contained more BoNT/A-specific antibody than did the aToxoid, and aBoNT/A was as effective as commercial rabbit antibody. The aToxoid provided no protection against BoNT/A in a standard mouse neutralization assay; however, 1 mg of PEG-extracted aBoNT/A neutralized 4,000 lethal doses of BoNT/A injected intraperitoneally. Based on these results, we calculated that in 1 month one hen could produce more than 100 liters of antibody detection reagents or enough antibody to neutralize approximately 11.6 million mouse lethal doses of botulinum toxin. Utilization of an egg antibody platform is potentially rapid (28 to 70 days) and scalable to kilogram quantities using current egg production facilities with as few as 1,000 hens.

Survival of Listeria monocytogenes in commercial cheese brines.

The survival of Listeria monocytogenes was determined in commercial cheese brines collected from cheese factories in Wisconsin and northern Illinois. Survival of L. monocytogenes inoculated into commercial cheese brines ranged from < 7 d to over 259 d. Survival did not correlate with pH, salt content, nitrogen content, mineral content, or inherent microbial populations but was negatively associated with addition of sodium hypochlorite at the dairy plant. The L. monocytogenes generally survived longer in brines held at 4 degrees C than at 12 degrees C. Sodium hypochlorite or hydrogen peroxide inactivated L. monocytogenes when added to commercial brines in the lab at 10 to 100 ppm or 0.001% to 0.02%, respectively. Addition of 1% potassium sorbate or 1% sodium benzoate also decreased survival of L. monocytogenes. Laboratory filtration of commercial brines had a negative effect on survival in one of three brines tested. The L. monocytogenes did not grow during incubation in any of the commercial brine samples tested.

Evaluation of botulinal toxin production in packaged fresh-cut cantaloupe and honeydew melons.

The ability of Clostridium botulinum to produce toxin on cubed, packaged melons was investigated relative to microbial spoilage at various incubation temperatures and in different packaging systems. Freshly cut cubes (approximately 2.5 cm3) of cantaloupe and honeydew melons were surface inoculated with a 10 strain mixture of proteolytic and nonproteolytic spores of C. botulinum (10 to 15 cubes per package; approximately 100 total spores per package). To initially evaluate toxin production and spoilage in a passively modified atmosphere, melon cubes were loosely packaged in air in polyethylene pouches, sealed, and incubated at 7 or 15 degrees C for up to 21 days. At various sampling intervals, samples were tested for headspace oxygen and carbon dioxide levels, pH, presence of botulinal toxin, aerobic and anaerobic plate counts, and counts of yeasts and molds. During incubation, headspace oxygen levels decreased, headspace carbon dioxide levels increased, aerobic and anaerobic plate counts increased, and the pH remained constant or decreased slightly. Botulinal toxin was not detected in any cantaloupe samples or in honeydew samples incubated at 7 degrees C. Botulinal toxin was detected in some honeydew samples at 15 degrees C after 9 days of incubation, but the toxic honeydews were severely spoiled and considered organoleptically unacceptable. A similar second experiment was performed in which half of the melon cubes were treated with UV light to inactivate vegetative organisms before packaging, and these were incubated at 7, 15, or 27 degrees C. In this second experiment, toxin production occurred in the UV-treated samples at 15 degrees C with gross spoilage and at 27 degrees C with only marginal spoilage. These data indicate that inhibition of spoilage organisms with UV light could result in botulinal toxin formation in packaged melons before overt spoilage.

A large outbreak of botulism: the hazardous baked potato.

In April 1994, the largest outbreak of botulism in the United States since 1978 occurred in El Paso, Texas. Thirty persons were affected; 4 required mechanical ventilation. All ate food from a Greek restaurant. The attack rate among persons who ate a potato-based dip was 86% (19/22) compared with 6% (11/176) among persons who did not eat the dip (relative risk [RR] = 13.8; 95% confidence interval [CI], 7.6-25.1). The attack rate among persons who ate an eggplant-based dip was 67% (6/9) compared with 13% (241189) among persons who did not (RR = 5.2; 95% CI, 2.9-9.5). Botulism toxin type A was detected from patients and in both dips. Toxin formation resulted from holding aluminum foil-wrapped baked potatoes at room temperature, apparently for several days, before they were used in the dips. Consumers should be informed of the potential hazards caused by holding foil-wrapped potatoes at ambient temperatures after cooking.

Antimicrobial activity of hop extracts against Listeria monocytogenes in media and in food.

Growth of Listeria monocytogenes was inhibited in culture media and in certain foods by four hop extracts (I-IV) containing varying concentrations of alpha-and beta-acids. Extracts (II and III) containing the highest concentrations of beta-acids were inhibitory at 0.01 mg/l in trypticase soy broth. In food, these hop extracts showed varying magnitudes of inhibition. In coleslaw, hop extract III at 1 mg/g enhanced the rate of inactivation of L. monocytogenes Scott A. Hop extract II was inhibitory at 0.1 and 1 mg/ml in skim and 2% milk, and was inhibitory at 1 mg/ml in whole milk. Hop extract II was listericidal in cottage cheese at 0.1 to 3 g/kg. No inhibition of L. monocytogenes by hop extract III was observed in Camembert cheese. Overall, the antimicrobial activity of hop extracts in food appeared to increase with acidity and lower fat content. Our results indicate that hop extracts could be used to control L. monocytogenes in minimally processed food with low fat content.

HEL and K562 cells: analysis of proteins by two-dimensional gel electrophoresis and comparison to erythroid and nonerythroid cells.

We have examined the total protein composition of the erythroleukemia cell lines K562 and HEL using high-resolution two-dimensional gel electrophoresis and have compared them with the pattern obtained by normal erythroid and nonerythroid cells. The proteins from the two cell lines, K562 and HEL, gave two-dimensional patterns that were similar to each other and to that of normal lymphocytes or leukemic cell lines. In contrast, normal erythroid precursor cells (BFUe-derived normoblasts) and erythrocytes have a protein profile that is characteristic and significantly different from that of normal lymphocytes or the leukemic cell lines examined. These data suggest a common protein profile in hemopoietic cells at early stages of differentiation (K562, HEL, or other leukemic lines) and in normal lymphocytes. Erythroid cells, in contrast to lymphoid cells, appear to diverge significantly from this common protein profile when differentiation proceeds to the level of morphologically recognizable erythroid cells. Induction of K562 and HEL cells by hemin produces changes in the abundance of several proteins, but fails to change the overall protein profile of the two cell lines.

Xylose isomerase from Escherichia coli. Characterization of the protein and the structural gene.

The gene that codes for xylose isomerase in Escherichia coli has been cloned by complementation of a xylose isomerase-negative E. coli mutant. The structural gene is 1320 nucleotides in length and codes for a protein of 440 amino acids. An additional 209 nucleotides 5' and 82 nucleotides 3' to the structural gene were also sequenced. To verify that the cloned gene encodes E. coli xylose isomerase, the enzyme was purified to homogeneity and the sequence of the first 25 amino acid residues was determined by a semimicromanual Edman procedure. These results establish that the NH2-terminal methionine of xylose isomerase is specified by an ATG which is 7 nucleotides downstream from a Shine-Dalgarno sequence.

Vitamin K-dependent carboxylase. Stoichiometry of carboxylation and vitamin K 2,3-epoxide formation.

Crude rat liver microsomal preparations catalyze a reduced vitamin K- and oxygen-dependent carboxylation of peptide-bound glutamyl residues to gamma-carboxyglutamyl residues. The same preparations convert reduced vitamin K to its 2,3-epoxide. The stoichiometry of this relationship has been investigated. At saturating concentrations of CO2, equal amounts of vitamin K 2,3-epoxide and gamma-carboxyglutamic acid are formed. As the CO2 concentration is lowered, this ratio shifts to a large excess of epoxide. Alterations in glutamyl substrate concentration or Mn2+ concentration cause equal alterations in both activities, while addition of KCN stimulated epoxidation and inhibited carboxylation. The release of 3H from a gamma-[3H]glutamyl substrate was tightly coupled to epoxide formation, and both of these activities were inhibited by glutathione peroxidase. These data are consistent with a reaction mechanism in which an oxygenated form of vitamin K activates the substrate glutamyl residue by hydrogen removal in a reaction that is coupled to vitamin K epoxide formation.

Vitamin K-dependent carboxylase: evidence for a hydroperoxide intermediate in the reaction.

Vitamin K is an essential cofactor for a microsomal carboxylase that converts glutamyl residues in endogenous precursor proteins to gamma-carboxyglutamyl residues in completed proteins. The same microsomal preparations convert vitamin K to its 2,3-epoxide, and it has been suggested that these two reactions (carboxylation and epoxidation) are coupled. Glutathione peroxidase, which reduces hydrogen peroxide and organic hydroperoxides, inhibits both of these reactions in a prepartion of microsomes solubilized by Triton X-100. Catalase has no effect. In the absence of vitamin K, and in the presence of NADPH, tert-butyl hydroperoxide acts as a weak vitamin K analog. At lower concentrations, tert-butyl hydroperoxide is an apparent competitive inhibitor of vitamin K for both the carboxylase and epoxidase reactions. These data are consistent with the hypothesis that both of these vitamin K-requiring reactions involve a common oxygenated intermediate, and that a hydroperoxide of the vitamin is the species involved.

Relationship between vitamin K-dependent carboxylation and vitamin K epoxidation.

It has been postulated that the liver microsomal conversion of vitamin K hydroquinone to its 2,3-epoxide (epoxidase activity) is coupled in some obligatory fashion to the vitamin K-dependent carboxylation (carboxylase activity) event also occurring in microsomes. This hypothesis is supported by the observations that the requirements for the two reactions are similar and that conditions that promote increased carboxylation increase the epoxidase activity. It has now been shown that both of these reactions are localized in the rough microsomal fraction of a cellular homogenate, and that both activities appear to be located on the luminal rather than the outer surface of microsomal membrane vesicles. The epoxidase activity has been found to be enriched as the microsomal carboxylase activity is fractionated, and a microsomal inhibitor of the carboxylase activity had been shown to also inhibit the epoxidase activity. The enzyme glutathione peroxidase inhibits both of these activities, suggesting that a hydroperoxide of the vitamin might be an intermediate for both reactions. The organic hydroperoxide t-butyl-OOH has also been shown to have weak vitamin K-like activity in an in vitro system. These data strengthen the hypothesis that these two reactions are related, perhaps through a common intermediate, but do not provide a definite molecular role for this interrelationship.