Steve L. Taylor, Ph.D. and Susan L. Hefle, Food Allergy Research & Resource Program, University of Nebraska–Lincoln

The field of food allergies and sensitivities has grown immensely in recent years. The presence of undeclared allergenic food residues in other foods is now one of the leading causes of recalls in the U.S. and Canada. Regulatory agencies in many countries are now allowing food manufacturers to use precautionary labelling terms such as "may contain peanuts" on food labels. Severe anaphylactic reactions to foods are now known to be more common than bee sting anaphylaxis. The Food Allergy Network in the U.S., the Anaphylaxis Network of Canada, and the Anaphylaxis Campaign in the U.K., among others, were formed to represent the interests of consumers. In the consumer response groups in major food companies, inquiries about food allergies and the composition of food products have increased rapidly. The food industry is now well aware of food allergies.

Food allergies afflict many more people in the population than once thought. A recent telephone survey at Mt. Sinai School of Medicine in New York indicates that 1.1% of Americans have peanut and/or tree nut allergies, with 0.6% having peanut allergy. A similar survey from the U.K. suggests that 0.5% of the British population have peanut allergies. Allergists seem to agree that the prevalence of food allergies, or at least severe food allergies, is increasing. The reasons for this increase are not understood.

These problems led to the creation of the Food Allergy Research & Resource Program (FARRP) at the University of Nebraska in 1995. The research programs of FARRP include: analyses to detect residues of allergenic foods that might contaminate other foods, the assessment of the allergenicity of various ingredients, the determination of threshold doses for allergenic foods, and the possible allergenicity of genetically modified foods.

The food industry needs analytical tools to assess the effectiveness of their cleaning operations when they use equipment that is shared between formulations that contain commonly allergic foods, and those that do not. ELISAs are now available for peanut, egg, casein, whey, almond, and soybean. The peanut and egg ELISAs have been converted into commercial kits by Neogen Corporation of Lansing, MI; the casein ELISA is soon to be released in a commercial kit format. Research is underway to develop ELISAs to the other tree nuts (walnut/pecan, hazelnut, cashew), clam, sesame seed, and shrimp. The assays are sensitive to the level of 2.5 ppm, sufficient to identify products that might be harmful to food-allergic consumers.

Many food ingredients are made from commonly allergenic sources. Casein and whey are obvious examples, Other such ingredients include peanut oil, soybean oil, various flavor formulations, and fish gelatin. Several clinical trials, including one conducted through FRI in 1981, have documented that highly refined peanut oil is safe for peanut-allergic individuals. Although one small trial was conducted on refined soybean oil through FRI in 1985, the number of patients involved in the trial has not been considered sufficient to prove the safety of soybean oil. Presently, another large clinical trial sponsored by the United Soybean Board is being initiated on safety of refined soybean oil for soybean-allergic individuals. This trial involves clinical collaborators from Univ. of Wisconsin, Medical College of Wisconsin, Univ. of Toronto, and Mt. Sinai School of Medicine.

How much is too much? That question is asked every time a company has a product containing undeclared residues of an allergenic food. Although it is reasonably well documented that food-allergic individuals can react to mere traces of the offending food, the exact thresholds are unknown. In a clinical trial conducted with the Univ. of Southampton in U.K., the threshold doses for peanuts were variable, with the most sensitive individual responding to only 2 mg of peanut. Currently, another clinical threshold experiment is underway on eggs at the Univ. of Arkansas Childrens Hospital and the Mt. Sinai School of Medicine.

Finally, concern has been expressed on the possible allergenicity of genetically modified foods. In actuality, the risk of allergenicity is quite small unless genes are transferred from known allergens. Several years ago we demonstrated that a transgenic soybean variety containing a gene from Brazil nut (to provide a methionine protein to deficient soybeans) expressed the allergenicity of Brazil nuts. Previously, the allergenicity of Brazil nuts was well documented but no one knew which Brazil nut protein was the allergen. It is important that this research was sponsored by Pioneer Hi-Bred International, the company that developed the transgenic soybean. When presented with the results, Pioneer abandoned any further commercial interest in this variety of soybeans. We view this episode as proof that the safety evaluation process for transgenic foods works.

• Contribution of dps to Acid- and Oxidative-stress Tolerance in Escherichia coli O157:H7
• CLA and Problems with Labeling Trans Fats
• Development of Formulation-Safe, Low Acid Foods
• Control of Trypanosomiasis 

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The dps gene of Escherichia coli O157:H7 (ATCC 43895) was cloned and sequenced. Comparison of the nucleotide sequence of dps from the serotype O157:H7 strain with E. coli K12 revealed two bp differences in the 504 nucleotides of the open reading frame; however, the nucleotide differences did not result in amino acid change. Five additional mismatches in the nucleotide sequences were identified upstream of the open reading frame, four occurred in a 12-bp region that was surrounded by a set of inverted repeats. Construction of a dps::nptI mutant in E. coli O157:H7 demonstrated that dps is involved in acid tolerance. Log-phase cells (5-h culture) of the dps mutant (FRIK 47991) challenged in synthetic gastric fluid (SGF, pH 1.8) for 3 h were significantly more sensitive (4-log₁₀ CFU/ml reduction) than the parent strain which decreased ca. 1.0-log₁₀ CFU/ml reduction.

Survival of early-stationary-phase cultures (12 h) of FRIK 47991 was similar to that observed with log-phase cells (ca. 4-log₁₀ CFU/ml reduction), while the numbers of the parent strain decreased approximately 2-log₁₀ CFU/ml after 3 h in SGF. In contrast, there was no difference in the survival of late-stationary-phase cells (24 h) of FRIK 47991 and the parent strain after 2 h of acid challenge in SGF. Log-phase cells of FRIK 47991 were also significantly more sensitive (ca. 4-log₁₀ CFU/ml reduction) to a 1-h challenge in phosphate-buffered saline containing 15 mM hydrogen peroxide than the parent strain (ca. 2.5-log₁₀ CFU/ml reduction). There was no significant difference in the survival of early- and late-stationary-phase cells of FRIK 47991 and the parent strain in hydrogen peroxide challenges. Complementation of FRIK 47991 (dps::nptI) with a functional dps restored acid and hydrogen peroxide tolerance to levels equal to or greater than those exhibited by the parent strain.

These results confirm the role of dps in protection from oxidative stress and establish its importance in acid tolerance of E. coli O157:H7. Further work will elucidate the regulation of this protein which may lead to strategies to control the tolerance properties of this pathogen.

Currently science and regulations have focused on a new category: "trans fatty acids." The Food and Drug Administration is proposing labeling of the amount of trans fatty acids contained in foods. Again the door is open for "reduced trans," "less trans" and "trans free" products in an attempt to eliminate the human diseases linked to these trans fatty acids that flourish in our population.

But trans fats are a category containing low levels of naturally occurring fatty acids, some of which have been shown experimentally to protect against human disease. For example, an 18-carbon fatty acid with a trans double bond at the 11th carbon is commonly found in milk fat. Bauman and coworkers at Cornell have shown that this fatty acid, when consumed, is converted by our body to a fatty acid now called cis 9, trans 11 conjugated linoleic acid (CLA). This fatty acid has been shown by Ip and coworkers at Roswell Cancer Institute in Buffalo to reduce the incidence of mammary carcinogenesis in rodents. In a study we published in 1994 we stated that we believe this trans fatty acid is a growth factor (for references see http://www.wisc.edu/cook). In addition, this trans fat and others have been shown in laboratory models to reduce and reverse atherosclerosis, increase bone mineral content, reduce body fat and increase lean mass, prevent body weight wasting associated with immune stress, yet enhance immune responsiveness and attenuate allergic type reactions. Categorical labeling can be misleading and dangerous to us all. If there is a specific compound that creates a risk of disease, it should be identified and labelled.

• The antilisterial effect of sodium lactate, sodium diacetate, or monolaurin were recently evaluated for several cured and uncured process meat products during refrigerated storage. Antimicrobials were used either in the product formulation or applied as a surface treatment after processing. In one study, Listeria monocytogenes did not grow on cured, smoked sausage formulated with 3.4% lactate and 1% diacetate stored for 12 weeks. This combination delayed but did not prevent pathogen growth on uncured, unsmoked sausage. Populations of L. monocytogenes increased on sausage formulated with <2% lactate and no diacetate. In a related study, wieners and hams were dipped for 5 seconds in antimicrobial solutions before inoculation with L. monocytogenes. Lactate/diacetate solutions were less effective when applied as a surface treatment than when used in product formulation. Dip solutions containing 6% lactate plus 3% diacetate delayed listerial growth on hams but not on wieners. Solutions containing 800 ppm monolaurin exhibited greater antilisterial activity than lactate/diacetate. Monolaurin prevented growth of L. monocytogenes on hams, whereas L. monocytogenes decreased >1-log₁₀ during the two-month storage on wieners treated with monolaurin. Additional research should be conducted to confirm these trends and to determine if the antilisterial effects can be enhanced using other combinations of antimicrobials.

• Bakery items (not cream-filled) traditionally have a short shelf-life and have not been considered potentially hazardous foods. Recently, modified atmosphere packaging has been introduced to extend shelf-life. We evaluated the botulinal stability of par-baked pizza crusts packaged under modified atmosphere and stored at 27ºC for eight months. Eight pizza crust formulations, having different combinations of pH and water activity (a_w), were inoculated with C. botulinum spores and assayed for the ability to support toxin production. Products formulated to pH <5.6 and a_w <0.94 did not support toxin production. Botulinal toxin was detected in products formulated to pH 6.0, a_w 0.94, or pH 5.9, a_w 0.95, indicating that pH and a_w are critical factors in the safety of bakery items under modified atmosphere. Manufactures should re-assess safety of their products when considering modified atmosphere packaging to extend shelf-life.

• Water activity and pH are also critical parameters in the control of growth of vegetative pathogens on process cheese slices. We previously reported that American-type process cheese slices formulated to a_w <0.93 and pH <5.8 did not support rapid and progressive growth of pathogens (J. Food Prot. 61:290–294), hence these formulations could be safely stored at ambient temperatures for at least 24 hours. Additionally, four process cheese food slice formulations, having higher pH and a_w values, were evaluated. Formulations included: (1) a_w O.945, pH 5.85, (2) a_w O.933, pH 5.9, (3) a_w O.940, pH 5.8, or (4) a_w 0.940, pH 5.5. As with the American-type slices, Salmonella, E. coli O157:H7, and L. monocytogenes survived, but did not grow on any formulation stored at 30ºC for 96 h. Staphylococcus aureus did not increase on slices with a_w <0.94 and pH <5.8. However, populations of S. aureus increased on slices with a_w 0.945, pH 5.85 and with a_w 0.933, pH 5.9 within 36 and 96 h, respectively. These data verify that process cheese safety is formula-specific and that slice formulations should be strictly controlled.

In a related publication (Cellular Immunology 192:24), we examined several macrophage-dependent mechanisms that might be activated by interferon-gamma to provide partial protection. Contrary to prior speculation from other labs, we showed that several biological characteristics associated with interferon-gamma activated macrophages were not linked to resistance, including nitric oxide production. Overall, the work continues to define the host-parasite interface in this deadly disease of man.

Byron Brehm-Stecher, M.S.,  Research Assistant	I am an alumnus of the UW–Madison, getting my M.S. in bacteriology with Professor Eric Johnson in 1996. Since then, I have been working with Dr. Johnson for my Ph.D. in two major areas: the development of novel antimicrobial strategies and rapid, nucleic acid-based detection of pathogenic bacteria using flow cytometry. For the first area, which stemmed from my Master's work, we have made important progress on the use of terpenoid compounds as enhancers of antimicrobial and antibiotic activity. We are in the later stages of seeking U.S. and foreign patents for this work, and I will be presenting some of our results this May at the general meeting of the American Society for Microbiology in Los Angeles. My Hatch-funded Ph.D. project is entitled "Rapid Detection of Salmonella by Flow Cytometry," and I have been working to achieve the goals of this grant for the past two and a half years. A recent highlight of this work has been the development of a method for substantially reducing the time needed for achieving bright, genus- or species-specific fluorescence in situ hybridization results. Additional work has focused on new methods for enhancing fluorescence in situ hybridization signals and on adapting existing means of cell capture for use in reducing enrichment times. We have recently been funded for a two-year project entitled "Rapid Nucleic Acid-based Detection and Enumeration of Listeria monocytogenes by Flow Cytometry." The ultimate goal of this and Salmonella work is to develop assays for the specific detection, enumeration and viability assessment of Salmonella sp. and L. monocytogenes in foods. Advantages of these assays will include the ability to detect fewer target cells than present rapid methods and the ability to detect small numbers of target cells against a high background of competing microflora. I'm originally from Silver Spring, Maryland, but have lived in Madison for the past eight years. I've been married seven years to my wife Susan, and we have two beautiful children, Emily (five) and Benjamin (two).
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I am currently a member of Dr. Wong's lab as a Ph.D. student, where I work on the project "Microbial Clogging of On-site Wastewater Disposal Systems." It has been three years since I came to the U.S.A. I was born in Seoul, Korea. I got a B.S degree from "Korea Univ." which is one of the most prestigious universities in Korea. In the U.S.A., I had worked as a master student in Dr. Tom Record's lab in the Biochemistry Department at Univ. of Wisconsin–Madison. The research I performed was "Macromolecular Crowding Effect on DNA-Protein Interaction by Using the Lac Operator-repressor System." Before coming to the U.S.A. I had a chance to study the resistance mechanism in transgenic plants against a plant virus at Kyoto University in Japan. With the background I have achieved, I would like to use biochemical and genetic approaches to elucidate the mechanism of microbial clogging in septic tanks. Our ultimate goal is to prevent on-site wastewater disposal systems from being clogged. We are now trying to use a transposon mutagenesis system for making "attachment deficient mutants" from environmentally isolated bacteria species. We hope that this study will be the primary step for understanding bacterial biofilm formation or microbial clogging in septic tanks. I joined Food Research Institute recently. I can say that people working here are very nice and generous to me. It was easy for me to adapt to the new environment within a short time.	Woojun Park, M.S.,  Research Assistant

The stated objective of the courses was "to assist individuals in developing the capability of evaluating the safety of heating and cooling procedures for meat and poultry products, and to improve understanding of relevant regulations related to such procedures."

Most sessions were held in the Department of Food Science, with hands-on process calculations in the computer classroom of the Biology New Media Center. Attendance was limited to 35, with both sessions full. Eleven papers and attendant discussions comprised the program, with speakers from industry and the University. Subjects included:
- Compliance guidelines — what they are and what they mean
- Fundamentals of microbiology
- Determining processing lethality
- Choosing your heating and cooling processes
- Process calculations — validating the safety of heating/cooling times and temperatures
- Equipment validation — making sure your equipment is doing what you expect it to do
- Strategies to control Listeria monocytogenes contamination in RTE products
- Panel: Dealing with deviations in heat processing and cooling (corrective action)

• The Rise and Fall of Salmonella enteritidis
• Mycobacterium paratuberculosis in Pasteurized Milk

One of these relatively new pests, Salmonella enteritidis, was once a relatively rare bacterium usually associated with rodents. Starting in the 1960s, chickens were identified as hosts for S. enteritidis, and human illness traced to infected poultry and eggs increased. At first the number of cases increased slowly, but by 1988, 15427 human cases of S. enteritidis were reported in England and Wales as compared to 6444 cases of S. typhimurium. Previously S. typhimurium cases always exceeded those of S. enteritidis. A similar rapid rise in cases was observed in the U.S. with similar numbers of S. typhimurium and S. enteritidis cases reported in 1989 in contrast to previous years with many fewer isolates of S. enteritidis. In 1997 the epidemic appeared to peak with 7,924 cases of S. enteritidis reported by CDC in the U.S. and 23,008 reported by the Public Health Laboratory Service in England.

Why and how did this former minor pathogen rise to such prominence? Recent research by Bäumler and coworkers (Science, Jan. 7, 2000, Vol. 287) traced the rise of S. enteritidis and demonstrated once again that tinkering with Mother Nature can have unexpected consequences. Since mice and rats carrying S. enteritidis forage for food around hen-houses, there is a ready source of infection for chickens, but this bacterium was rarely detected in chickens before the 1960s. However, prior to the 1970s, chicken flocks were often infected with S. pullorum and S. gallinarum, two species causing serious illness in poultry and economic losses to the poultry industry. Both England and the U.S. undertook aggressive campaigns to rid chicken flocks of these pathogens and largely succeeded by the mid 1970s. With the demise of these bacteria, an ecological niche was opened up, and S. enteritidis took advantage of it.

Salmonella enteritidis was able to thrive with the eradication of S. pullorum and S. gallinarum because all three serotypes have similar O9 antigens containing a tyvelose sugar. Thus chickens infected with the pathogenic species produced antibodies which also acted against S. enteritidis. When these two pathogens were eradicated, then S. enteritidis was free to infect chickens although it did not cause illness in the birds. (S. typhimurium has different sugars in its polysaccharide layer and its antigens do not cross-react with O9. Therefore S. typhimurium populations were not affected by the disappearance of S. pullorum and S. gallinarum.)

A look at very recent data on the incidence of S. enteritidis infections in humans reveals a distinct downward turn since 1997. Cases appeared to peak in England in 1997 at 23,008 and then declined to 16,196 in 1998 and approximately 8,600 in 1999. Cases in the U.S. declined from 7,924 in 1997 to 5,900 in 1998. While 1999 data are not complete yet, it appears that a further significant decline occurred last year. As yet we have no definite explanation for this fall in numbers of S. enteritidis. Most likely, both greater consumer awareness of the importance of cooking eggs properly and greater efforts on the part of the poultry industry to produce cleaner birds and eggs have contributed to the decrease.

Many features of Crohn's Disease in humans resemble those of Johne's Disease in cattle and numerous research groups have attempted to isolate M. paratuberculosis from lesions in the gut of Crohn's patients or to identify antibodies to M. paratuberculosis in the blood of these patients. Both positive and negative results have been reported. The latest research, published in 1999, included: (1) data showing that serum IgE antibodies from patients with Crohn's Disease had a significantly greater reaction to M. paratuberculosis than IgE from healthy persons or from patients with ulcerative colitis or tuberculosis and (2) a review which concluded that there was not as yet definitive evidence for the involvement of M. paratuberculosis in Crohn's Disease. Even if M. paratuberculosis is found to be associated with Crohn's Disease, there is still the question whether it causes this disease, though the similarities between Crohn's and Johne's diseases are suggestive of a connection.

Recent experimental work (1999) has demonstrated that milk contaminated with 10⁶ cfu M. paratuberculosis/ml is not reliably inactivated by current high-temperature short-time pasteurization conditions (72ºC, 15 sec). Exposure to higher temperatures (75–90ºC) for 15 sec likewise allowed survival of some M. paratuberculosis cells even though there was a 5-6 log₁₀ kill; a longer exposure (25 sec) to 72ºC did completely inactivate high concentrations of M. paratuberculosis. It appears that clumping of cells helps to protect those in the interior by retarding heat transfer so that the internal cells are not actually exposed to high enough temperatures. Therefore, to insure sterilization of milk as regards M. paratuberculosis, it may be necessary to increase the holding time and/or temperature.

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