The worm Adult Activity Test (wAAT): A de novo mathematical model for detecting acute chemical effects in Caenorhabditis elegans.

We have adapted a semiautomated method for tracking Caenorhabditis elegans spontaneous locomotor activity into a quantifiable assay by developing a sophisticated method for analyzing the time course of measured activity. The 16-h worm Adult Activity Test (wAAT) can be used to measure C. elegans activity levels for efficient screening for pharmacological and toxicity-induced effects. As with any apical endpoint assay, the wAAT is mode of action agnostic, allowing for detection of effects from a broad spectrum of response pathways. With caffeine as a model mild stimulant, the wAAT showed transient hyperactivity followed by reversion to baseline. Mercury chloride (HgCl2 ) produced an early dose-response hyperactivity phase followed by pronounced hypoactivity, a behavior pattern we have termed a toxicant "escape response." Methylmercury chloride (meHgCl) produced a similar pattern to HgCl2 , but at much lower concentrations, a weaker hyperactivity response, and more pronounced hypoactivity. Sodium arsenite (NaAsO2 ) and dimethylarsinic acid (DMA) induced hypoactivity at high concentrations. Acute toxicity, as measured by hypoactivity in C. elegans adults, was ranked: meHgCl > HgCl2 > NaAsO2 = DMA. Caffeine was not toxic with the wAAT at tested concentrations. Methods for conducting the wAAT are described, along with instructions for preparing C. elegans Habitation Medium, a liquid nutrient medium that allows for developmental timing equivalent to that found with C. elegans grown on agar with OP50 Escherichia coli feeder cultures. A de novo mathematical parametric model for adult C. elegans activity and the application of this model in ranking exposure toxicity are presented.

Diet-induced obesity precipitates kidney dysfunction and alters inflammatory mediators in mice treated with Shiga Toxin 2.

Shiga Toxin (Stx)-producing E. coli (STEC) continue to be a prominent cause of foodborne outbreaks of hemorrhagic colitis worldwide, and can result in life-threatening diseases, including hemolytic uremic syndrome (HUS), in susceptible individuals. Obesity-associated immune dysfunction has been shown to be a risk factor for infectious diseases, although few studies have addressed the role of obesity in foodborne diseases. We hypothesized that obesity may affect the development of HUS through an alteration of immune responses and kidney function. We combined diet-induced obese (DIO) and HUS mouse models to look for differences in disease outcome between DIO and wild-type (WT) male and female C57 B l/6 mice. Following multiple intraperitoneal injections with endotoxin-free saline or sublethal doses of purified Stx2, we examined DIO and WT mice for signs of HUS development. DIO mice receiving Stx2 injections lost more body weight, and had significantly higher (p < 0.001) BUN, serum creatinine, and neutrophil counts compared to WT mice or DIO mice receiving saline injections. Lymphocyte counts were significantly (p < 0.05) lower in Stx2-treated obese mice compared to WT mice or saline-treated DIO mice. In addition to increased Stx2-induced kidney dysfunction, DIO mouse kidneys also had significantly increased expression of IL-1α, IL-1ß, IL-6, TNF-α, MCP-1, and KC RNA compared to saline controls (p < 0.05). Serum cytokine levels of IL-6 and KC were also significantly higher in Stx2-treated mice compared to saline controls, but there were no significant differences between the WT and DIO mice. WT and DIO mice treated with Stx2 exhibited significantly higher degrees of kidney tubular dilation and necrosis as well as some signs of tissue repair/regeneration, but did not appear to progress to the full pathology typically associated with human HUS. Although the combined obesity/HUS mouse model did not manifest into HUS symptoms and pathogenesis, these data demonstrate that obesity alters kidney function, inflammatory cells and cytokine production in response to Stx2, and may play a role in HUS severity in a susceptible model of infection.

Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells.

A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1 % dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2 weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 >> HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC 50 within 2.5-fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity.

Listeria monocytogenes Contamination Leads to Survival and Growth During Enoki Mushroom Cultivation.

Two recent outbreaks of listeriosis have been linked to the consumption of enoki mushrooms. After the first outbreak, import sampling by the U.S. FDA identified that 43% of the samples evaluated were positive for Listeria monocytogenes (Lm). These observations raised questions about the potential sources of Lm contamination of enoki mushrooms. One potential source of contamination is during enoki mushroom cultivation, as growing conditions are comparatively cool and moist to induce mushroom germination, to which Lm is well adapted. Two varieties of enoki mushrooms were evaluated to determine the potential for Lm to contaminate enoki cultures when introduced at various points during cultivation (inoculation, scraping, pinning, and collaring). The results of two trials showed that Lm established contamination and grew to similar levels in the substrate regardless of when Lm was introduced and, with one exception, did not alter the rate of mushroom generation to below the control. Enumeration of Lm in enoki mushroom cultures at harvest found an average contamination of 103 cfu/g, though the results were variable. Refrigerated storage for six weeks was found to result in an increase in Lm. Additionally, no statistically significant difference in the levels of Lm was observed based on proximity to the substrate, though levels of Lm in the different enoki samples correlated with levels of Lm in the substrate at harvest, but not at scraping. The ability of Lm to grow independently in the media used to culture enoki was assessed, and Lm was found to be unable to grow but could sporadically survive in Masters Mix. No growth of Lm was observed in potato dextrose broth, though growth could occur on the agar. Overall, the data indicate a high potential for the establishment of Lm contamination at any point during enoki cultivation to result in Lm-contaminated mushrooms. These data indicate a need for active control mechanisms to prevent the introduction of Lm to enoki cultures.

Detection Limits of Insect Fragments in Spiked Whole Wheat Flour Using Multiplex Polymerase Chain Reaction (PCR).

The need for a sensitive molecular method to detect specific species of insect contaminants in food products remains a significant challenge in the food industry. This study evaluated the detection limit of a multiplex end-point PCR assay for detecting insects in food. The assay amplifies two fragments of the cytochrome oxidase subunit I gene (COI-Fa and COI-Fb) and one fragment of the protein-coding wingless (wg) gene found in insects. Five insect species, comprising three vectors of foodborne pathogens (the housefly, Musca domestica, the American cockroach, Periplaneta americana, and the pharaoh ant, Monomorium pharaonis) and two storage insect pests (the red flour beetle, Tribolium castaneum and the Indian meal moth, Plodia interpunctella), were spiked separately and in combination at levels of 1, 0.1, 0.01, and 0.001% in whole wheat flour. At spike levels greater than 0.01%, amplicon bands of expected sizes were seen in 100% of samples containing fragments from distinct insect species. At least 25% of spiked samples at the lowest spike level had amplicon bands, except for samples spiked with M. domestica. Results showed a 18.9% probability (with 11.3% and 30% lower and upper confidence limits, respectively) of detecting insect fragments at the lowest spike level (0.001%, corresponding to 3-22 fragments), which is far below the FDA's regulatory level of less than 75 fragments per 50 g of wheat flour. The intensity of amplicon bands in the gel images was higher at higher spike levels. However, this method is not quantitative enough to extrapolate the intensity of the amplicon bands to the number of insect fragments present in a sample. This multiplex assay was also evaluated in a variety of market food samples derived from plants and animals, showing its potential use in various food types. Overall, the sensitivity and specificity of this molecular approach suggest that it could be used in the future as a screening tool for detecting insect contaminants in food.

Persistence comparison of two Shiga-toxin producing Escherichia coli (STEC) serovars during long-term storage and thermal inactivation in various wheat flours.

Foodborne outbreaks associated with Shiga toxin-producing Escherichia coli (STEC) contaminated wheat flour have been an increasing food safety concern in recent decades. However, there is little literature aimed at investigating the impact of different flour types on the persistence of STEC during storage and thermal inactivation. Therefore, two serovars of STEC, O121 and O157, were selected to inoculate each of five different types of common wheat flours: whole wheat, bleached, unbleached, bread, and self-rising. Inoculated flours were examined for the stability of STEC during storage for up to 42 days at room temperature (RT) and aw ~0.56. Additionally, the thermal resistance of O121 and O157 under isothermal conditions at 60, 70, 80, and 90°C was analyzed for the inoculated flours. STEC storage persistence at RT was generally not affected by flour type, however, decreases of 1.2 and 2.4 log CFU/day within whole wheat flour for O121 and O157, respectively, were significantly lower than other flours. Though few differences were identified in relation to flour type, O121 exhibited significantly better survival rates than O157 during both equilibrium and storage periods. Compared to an approximate 6 log reduction in the population of O157, O121 population levels were reduced by a significantly lower amount (~3 log) during the entire storage period at RT. At each isothermal temperature, the impact of flour type on the thermal resistance capabilities of O121 or O157 was not a significant factor and resulted in similar survival curves regardless of serovar. Instead of exhibiting linear survival curves, both O121 and O157 displayed nonlinear curves with some shoulder/tail effect. Similar for both O121 and O157, the predicted decimal reduction time (D-value) decreased from approximately 25 min to around 8 min as the isothermal temperature increased from 60°C to 90°C. Results reported here can contribute to risk assessment models concerning contamination of STEC in wheat flour and add to our understanding of the impacts of flour type and STEC serovar on desiccation stability during storage and isothermal inactivation during thermal treatment.

Multilab Validation Report for the Verification and Subtyping of Listeria monocytogenes Using qPCR.

Listeria monocytogenes (Lm) is a Gram-positive bacterium that causes invasive listeriosis, an illness with high mortality and hospitalization rates. Due to the severity of illness associated with Lm, rapid identification and characterization of isolates from foods and the food-processing environment are critical to properly identify and track the pathogen and quickly remove adulterated foods from the market. Prior methods can rely on time-consuming biochemical or sera-agglutination assays to perform these tasks. Development of a high-throughput method that would rapidly perform these tasks is critical to improve response to contamination events. Previously, a single laboratory validation of a qPCR-based method was presented that could rapidly verify Lm isolates and characterize them into six molecular serogroups. In the current study, a multi-laboratory validation (MLV) was performed to evaluate the reliability of the qPCR method for identification and serogrouping of Lm isolates. Sixteen collaborating laboratories independently analyzed a panel of 43 blinded isolates plus three control strains using the qPCR method. This panel was comprised of representatives for non-Listeria (n = 7), Listeria sp. (n = 8), and Lm (n = 28) strains. The Lm isolates contained representatives of the six serogroups: 2A, 2B, 2C, 4B, NT, and 4bV/IVb-v1, with five strains for each serogroup except 4bV/IVb-v1 (n = 3). The results generated by 16 laboratories showed high sensitivity, specificity, and accuracy, generally ≥97%, for both the genus-species and serogrouping qPCRs. Results from one laboratory lowered the sensitivity of the non-Listeria group to 93%. These results indicated the method was highly reliable. However, only the previously evaluated serogroups were tested within the MLV panel, though there is the potential for other serogroup results. Sequence Read Archive (SRA) files for Lm isolates were evaluated to determine the frequency of other potential serogroup profiles. This effort identified a low percentage of isolates with atypical qPCR serogroups (0.30%) that are consistent with Lm and were generally associated with lineage II and the natural environment. In summary, the results indicate that the proposed qPCR method is reliable and has a high degree of sensitivity, accuracy, and specificity, while also decreasing hands-on analysis time and increasing throughput of the analysis.

A longitudinal study to examine the influence of farming practices and environmental factors on pathogen prevalence using structural equation modeling.

The contamination of fresh produce with foodborne pathogens has been an on-going concern with outbreaks linked to these commodities. Evaluation of farm practices, such as use of manure, irrigation water source, and other factors that could influence pathogen prevalence in the farming environment could lead to improved mitigation strategies to reduce the potential for contamination events. Soil, water, manure, and compost were sampled from farms in Ohio and Georgia to identify the prevalence of Salmonella, Listeria monocytogenes (Lm), Campylobacter, and Shiga-toxin-producing Escherichia coli (STEC), as well as Arcobacter, an emerging human pathogen. This study investigated agricultural practices to determine which influenced pathogen prevalence, i.e., the percent positive samples. These efforts identified a low prevalence of Salmonella, STEC, and Campylobacter in soil and water (< 10%), preventing statistical modeling of these pathogens. However, Lm and Arcobacter were found in soil (13 and 7%, respectively), manure (49 and 32%, respectively), and water samples (18 and 39%, respectively) at a comparatively higher prevalence, suggesting different dynamics are involved in their survival in the farm environment. Lm and Arcobacter prevalence data, soil chemical characteristics, as well as farm practices and weather, were analyzed using structural equation modeling to identify which factors play a role, directly or indirectly, on the prevalence of these pathogens. These analyses identified an association between pathogen prevalence and weather, as well as biological soil amendments of animal origin. Increasing air temperature increased Arcobacter and decreased Lm. Lm prevalence was found to be inversely correlated with the use of surface water for irrigation, despite a high Lm prevalence in surface water suggesting other factors may play a role. Furthermore, Lm prevalence increased when the microbiome's Simpson's Diversity Index decreased, which occurred as soil fertility increased, leading to an indirect positive effect for soil fertility on Lm prevalence. These results suggest that pathogen, environment, and farm management practices, in addition to produce commodities, all need to be considered when developing mitigation strategies. The prevalence of Arcobacter and Lm versus the other pathogens suggests that multiple mitigation strategies may need to be employed to control these pathogens.

Evaluation of the utility of the Beta Human Liver Emulation System (BHLES) for CFSAN's regulatory toxicology program.

Microphysiological systems (MPS), such as organ-on-a-chip platforms, are an emerging alternative model that may be useful for predicting human physiology and/or toxicity. Due to the interest in these platforms, the Center for Food Safety and Applied Nutrition partnered with Emulate to evaluate the utility of the Beta Human Liver Emulation System (BHLES) for its regulatory science program. Using known hepatotoxic compounds (usnic acid, benzbromarone, tamoxifen, and acetaminophen) and compounds that have no reported human cases of liver toxicity (dimethyl sulfoxide, theophylline, and aminohippurate) the platform's performance was evaluated. Chemical toxicity was assessed by albumin secretion, urea and LDH release, nuclei number, mitochondrial membrane potential, and apoptosis. System/platform performance was evaluated in terms of sensitivity and specificity, power, and variability and repeatability. Chemical interactions with the Chip material were also assessed. Preliminary findings suggested that for the model test compounds selected, the BHLES accurately predicted toxicity, demonstrated high sensitivity and specificity, high power, and low variability. However, some compounds interacted with the Chip material indicating variable exposure levels that should be accounted for when planning experimentation. The details of the evaluation are presented herein.

Characteristics of U.S. Consumers Reporting Past Year Intake of Raw (Unpasteurized) Milk: Results from the 2016 Food Safety Survey and 2019 Food Safety and Nutrition Survey.

ABSTRACT: Consumption of unpasteurized (raw) milk has been linked to foodborne illness in the United States at higher relative rates than has consumption of pasteurized milk and milk products. Regulation of these products differs by state. Regardless of the risk of consumption, some people still purchase and consume unpasteurized milk. Based on information from the 2016 Food Safety Survey and the 2019 Food Safety and Nutrition Survey conducted by the U.S. Food and Drug Administration, we evaluated prevalence, frequency, and demographic predictors of consumption of raw milk in the U.S. adult population. Results show that 4.4% of U.S. adults reported consuming raw milk at least once in the past year, with 1.6% reporting frequent consumption of raw milk (once per month or more often) and 1.0% reporting consumption once per week or more often. The individuals who consumed raw milk in the previous 12 months were more likely to be younger, living in a rural area, and living in a state in which retail sale of raw milk is legal. These results provide quantitative information on consumption prevalence and frequency and demographic characteristics of individuals who consume unpasteurized milk in the United States.

Health Disparities in Calorie Knowledge and Confidence Among the U.S. Adult Population.

PURPOSE: Obesity prevalence has reached an all-time high in the US, affecting >40% of the population. This study's objective was to evaluate associations between demographics and self-reported calorie knowledge and self-perceived confidence in calorie knowledge ("calorie confidence"). The relationships between body mass index (BMI) and calorie knowledge and confidence were also explored. METHODS: We analyzed data from participants (n = 2171) in the crosssectional, nationally representative 2019 FDA Food Safety and Nutrition Survey using logistic regression to estimate adjusted odds ratios (AORs) and confidence intervals (95% CIs) for associations between BMI and calorie knowledge (correct/incorrect), calorie confidence (confident/not confident), and demographic characteristics, and the Wald chi square test to evaluate relationships between BMI and both calorie knowledge and confidence. RESULTS: Many of the same subgroups were more likely than others to report lack of calorie knowledge and lack of confidence in knowing the typical daily calorie intake needed to maintain a healthy weight [respective AORs (95% CIs): age (years), >60 vs 51-60, 1.7 (1.1-2.5), and 1.4 (1.0-2.0); sex, male vs female, 1.7 (1.3-2.3), and 1.7 (1.3-2.1); race/ethnicity, non-Hispanic Black vs non-Hispanic white, 3.4 (2.1-5.5), and 2.4 (1.6-3.8); education, ≤high school vs college grad, 1.5 (1.0- 2.3), and 1.9 (1.3-2.7)]. BMI was significantly correlated with calorie confidence (P = .047), such that those reporting less confidence were more likely overweight or obese [underweight/healthy (BMI < 25): 29%, overweight (25 ≤ BMI < 30): 34%, obese (BMI ≥ 30): 37%]. CONCLUSION: In certain demographic subgroups associations between calorie knowledge and confidence differed. Tailored education and outreach for these groups may help to address these disparities.

Development and Validation of a Quantitative PCR Method for Species Verification and Serogroup Determination of Listeria monocytogenes Isolates.

ABSTRACT: Listeria monocytogenes (Lm) is one of the leading causes of death because of foodborne illness, affecting the elderly, pregnant women, neonates, and people who are immunocompromised. Serologically, Lm can be classified into 13 serotypes, although only 4 are typically linked with food contamination and illness. Since 2000, a shift in serotypes involved in listeriosis outbreaks has been observed, suggesting that tracking of serotypes could help identify emerging trends. A PCR method developed in 2004 allowed detection of the four major serotypes as molecular serogroups, corresponding to broad phylogenetic groups. In this study, a novel quantitative PCR (qPCR) method was developed that uses two multiplex qPCRs, one to confirm the Listeria genus and Lm species and the second for Lm molecular serogrouping. This method was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) method for Lm and the seroagglutination method, using a 208-strain panel. Comparison of the genus and species qPCR assay with the BAM methods found an equal or slightly higher accuracy for the qPCR method (>98%), compared with the BAM protocol (>96%), when evaluated against independent characterization data. Molecular serogrouping using the qPCR method (96.6%) was more accurate than the seroagglutination assay (75.6%). The qPCR method identified Lm 4bV strains, which could not be resolved using seroagglutination. The qPCR could not identify lineage III and IV serotype 4b strains but did correctly identify 16 of 18 lineage III and IV strains. The qPCR method performed genus identification for the Listeria species Lm, L. innocua, L. welshimeri, L. ivanovii, and L. seeligeri. In addition, the method performed species identification for Lm and classified Lm into six molecular serogroups: 2A, 2B, 2C, 4B, NT, and 4bV. This method provided a rapid and accurate confirmation of Lm and serogroup determinations; furthermore, it could help identify otherwise unlinked strains by enabling whole genome sequencing analysis based on broad phylogeny, independent of other information.

Virulence assessment of Listeria monocytogenes grown in different foods using a Galleria mellonella model.

Various produce including cantaloupe, caramel-coated apples, and packaged salads, have been recognized in recent years as vehicles for listeriosis, a human foodborne disease caused by intracellular pathogen Listeria monocytogenes. Our knowledge regarding the role of these foods in L. monocytogenes virulence, however, is limited. Understanding their role in modulating L. monocytogenes virulence can be useful in risk assessments and for developing control measures. In this study, we employed the Galleria mellonella larvae model to evaluate virulence potential of fifteen clinical, environmental and food isolates of L. monocytogenes, related to three major outbreaks, after growth on different foods. The non-human pathogen Listeria innocua was also included in the panel. Strains were inoculated in parallel in 5ml of brain heart infusion (BHI) broth, and on the surfaces of cantaloupe and apple fragments (5g each) at about 105 colony forming units (CFU)/ml/fragment. One set of inoculated broth and food fragments was incubated at 10°C for 5 days while the second set was kept at 25°C for 3 days. L. monocytogenes cells were recovered from the fruits and BHI, washed twice, re-suspended in saline, and used to inoculate G. mellonella larvae at final concentrations of 106 and 105 CFU/larva. The larvae were incubated at 37°C and monitored for mortality (LT50-time taken to kill 50% of the larvae) and phenotypic changes over seven days. L. monocytogenes grown on cantaloupe and apple flesh surfaces resulted in higher virulence than when grown in BHI. L. monocytogenes infection at 106 CFU/larvae resulted in an average LT50 of ≤ 30, 36 and 47 hours on cantaloupe, apples and BHI, respectively. These results represent a 2.5-4-fold increased mortality compared with an LT50 ≥120 hours in larvae infected with the same doses of L. innocua grown in corresponding matrices. Similar trends were also recorded with doses of about 105 CFU /larvae.

Utilization of a model hepatotoxic compound, diglycolic acid, to evaluate liver Organ-Chip performance and in vitro to in vivo concordance.

Microphysiological systems (MPS) are emerging as potentially predictive models for drug safety and toxicity assessment. To assess the utility of these systems, the Food and Drug Administration partnered with Emulate to evaluate the Human Liver Organ-Chip in a regulatory setting. Diglycolic acid (DGA), a known hepatotoxin, was evaluated in the Liver-Chip and compared to a multi-well plate format to assess the Liver-Chip's capabilities, limitations, overall performance, and concordance with other in vivo and in vitro studies. Cryopreserved primary human hepatocytes were exposed to DGA from 1 to 20 mM in Liver-Chips or traditional multi-well plates. We found that 10 mM or 20 mM of DGA was severely cytotoxic in both platforms, while 5 mM was mildly cytotoxic in Liver-Chips. Additionally, some hepatocyte functions were reduced with 5 mM DGA in Liver-Chips and 1 mM in well plates. Individual well effects were greater or occurred sooner than in the Liver-Chips. Examination of the performance of the Liver-Chip showed that variability was low for biochemical endpoints, but higher for imaging endpoints. Sensitivity and specificity were high. Only 3-4 Liver-Chips were necessary to detect an effect depending on the endpoint and effect size. The specifics of the experiment are found herein.

Analysis of food-allergic and anaphylactic events in the National Electronic Injury Surveillance System.

BACKGROUND: The National Electronic Injury Surveillance System (NEISS) captures a nationally representative probability sample from hospital emergency departments (EDs) in the United States. OBJECTIVE: Emergency department data from NEISS were analyzed to assess the magnitude and severity of adverse events attributable to food allergies. METHODS: Emergency department events describing food-related allergic symptomatology were identified from 34 participating EDs from August 1 to September 30, 2003. RESULTS: Extrapolation of NEISS event data predicts a total of 20,821 hospital ED visits, 2333 visits for anaphylaxis, and 520 hospitalizations caused by food allergy in the United States during the 2-month study period. The median age was 26 years; 24% of visits involved children < or =5 years old. Shellfish was the most frequently implicated food in persons > or =6 years old, whereas children < or =5 years old experienced more events from eggs, fruit, peanuts, and tree nuts. There were no reported deaths. Review of medical records found that only 19% of patients received epinephrine, and, using criteria established by a 2005 anaphylaxis symposium, 57% of likely anaphylactic events did not have an ED diagnosis of anaphylaxis. CONCLUSION: Analysis of NEISS data may be a useful tool for assessing the magnitude and severity of food-allergic events. A criteria-based review of medical records suggests underdiagnosis of anaphylactic events in EDs.

Extraction Conditions Affect the Immunoreactivity of Peanut Allergens.

Peanut allergic consumers rely on food package labels to avoid foods containing peanut. The inadvertent presence of peanut in foods due to cross-contact can be fatal if ingestion of such food leads to an allergic reaction. Analytical methods are available to detect undeclared peanut in foods. However, depending on the type of food matrix and food processing parameters, method performance can be adversely affected due to reduction in the extraction efficiency of peanut proteins. Temperature and probe sonication were used as a preincubation treatment for peanut flour slurries to assess their effect on the total peanut protein solubility from raw, light-roasted, and dark-roasted peanut flours. The effect of these treatments on the immunoreactivity of peanut allergens (Ara h 1, Ara h 2, Ara h 3, and Ara h 6) was determined by an indirect enzyme-linked immunosorbent assay using antibodies raised against these individual peanut proteins. Preincubation at 50 °C did not significantly improve the peanut protein solubility, whereas an increase in protein solubility was observed when light- and dark-roasted peanut flour slurries were preincubated at 90 °C or sonicated. The immunoreactivity of peanut allergens varied depending on the degree of peanut flour roasting and type of preincubation treatment. Overall, the immunoreactivity of peanut allergens from most peanut flour slurries was unaffected when preincubated at 50 °C for up to 60 min or sonicated with a probe for up to 5 min, whereas preincubation at 90 °C resulted in a time-dependent reduction in immunoreactivity of peanut allergens. Sonication treatment may improve peanut protein extraction without markedly affecting their immunoreactivity. PRACTICAL APPLICATION: Extraction of peanut proteins is vital for developed analytical methods to estimate peanut allergens in foods. The manuscript describes the effect of two different temperatures (50 and 90 °C) and probe-type sonication on peanut protein solubility. The findings suggest sonication can improve peanut protein solubility without markedly affecting their immunoreactivity.

Enhanced quantitation of egg allergen in foods using incurred standards and antibodies against processed egg in a model ELISA.

Underestimation of egg allergen from processed foods prompted the evaluation of critical Enzyme-Linked Immunosorbent Assay (ELISA) parameters: (1) extraction of egg proteins from a processed matrix; (2) use of anti-heat processed egg antibodies (Abs) on detectability of modified proteins, and (3) utilization of incurred material as standards. The relative affinity of two combinations of raw (R), boiled (B) and fried (F) Abs to unprocessed/processed egg proteins with or without matrix was determined from antibody (Ab) binding curves. In ELISAs using RBF-Abs and BF-Abs, denaturing buffer, and incurred standards, the Limit of Detection (LOD) and Limit of Quantitation (LOQ) were 0.47 and 0.25; and 1.58 and 0.85, respectively, and the linear range was 0-24 µg g-1 egg protein. The recoveries of egg protein from cookies, cereal bar, and muffin (incurred levels 4.8-48 µg g-1) with the developed ELISAs were in an acceptable range (50-130%). These ELISAs consistently detected more declared/undeclared egg proteins in market samples compared to assays using PBS for extraction. Overall, better assay performance was observed using BF-Abs. An ELISA combining anti-processed egg Abs, denaturing buffer, and incurred standards promises improved quantitation of egg proteins in processed foods.

Reported Knowledge of Typical Daily Calorie Requirements: Relationship to Demographic Characteristics in US Adults.

BACKGROUND: US obesity rates are at historically high levels, increasing the risk of negative health and economic outcomes at individual and population levels. Findings from earlier studies indicate that many consumers lack a clear understanding of calorie needs, potentially affecting their ability to manage caloric intake. OBJECTIVE: Our aim was to determine the knowledge of typical daily calorie needs of US adults by demographic and other characteristics, using a nationally representative sample. DESIGN: Data were analyzed from 6,267 respondents to the 2007-2008 and 2009-2010 National Health and Nutrition Examination Survey and its supplemental data source, the Flexible Consumer Behavior Survey, to assess reported knowledge of typical daily calorie requirements and associations with demographic and other characteristics of interest. STATISTICAL ANALYSES PERFORMED: Logistic regression for complex sample surveys was used to estimate associations between self-reported daily calorie needs for men and women aged 21 years and older and participant characteristics. RESULTS: Most respondents accurately reported typical daily calorie needs for a person of their sex, age group, and physical activity level, however, distinct differences emerged between demographic groups. Women, non-Hispanic whites, and those with higher income and education levels were more likely to estimate typical daily calorie needs accurately; men were almost four times more likely than women to indicate a lack of knowledge of daily calorie needs. CONCLUSIONS: Knowledge of typical daily calorie requirements is a foundational concept of nutrition literacy. Educational efforts to increase awareness, knowledge, and use of calorie information for certain groups may be helpful to refine interventions and ultimately improve public health in the United States.

Multiendpoint mechanistic profiling of hepatotoxicants in HepG2/C3A human hepatoma cells and novel statistical approaches for development of a prediction model for acute hepatotoxicity.

HepG2/C3A human hepatoma cells were exposed to serial concentrations of seven known hepatotoxicants for 48h. Six endpoint assays were selected to model different mechanisms of acute hepatotoxicity. Each compound produced a unique concentration-response pattern across all endpoints. The endpoints did not correlate strongly, suggesting that each endpoint monitored an independent cellular process. Prediction models were developed using five statistical methods. The models used only known hepatotoxicants for the training set. The zero concentration (control) and all concentrations not significantly different from control were programmed as non-toxic levels and concentrations significantly different from control as toxic levels. So, rather than a binary classification of each compound (i.e., toxic or non-toxic), the models gave a prediction of the concentration, if any, at which a compound showed behavior similar to liver toxicants at their toxic concentrations. The discriminant analysis model gave the best overall performance with positive and negative predictive values of 1.00 and 0.83, respectively. Ten additional compounds were tested using this prediction model. The model predicted liver active concentrations for each compound that were consistent with their known biologically active concentrations. This model system may be useful for predicting concentration levels at which unknown compounds would display undesirable liver activity.

Silver acetate exposure: Effects on reproduction and post natal development.

Effects of oral silver acetate exposure were assessed in P generation and F generation post-natal day 26 rats. Male and female Sprague Dawley rats (n = 20 each) were exposed to silver acetate at 0.4, 4.0 or 40.0 mg/kg bw in their drinking water for 10 weeks prior to and during mating. Females were exposed to silver acetate throughout gestation and lactation. Clinical signs, body weight, feed and fluid consumption were recorded regularly. Decreased mean daily fluid consumption was observed in male and female animals during the 10 week pre mating period and during gestation in the 40 mg/kg bw dose group. Decreased fertility was observed in the 40 mg/kg bw dose group. Decreased feed consumption was observed across all dose groups and decreased mean daily fluid consumption was observed in the 4.0 mg/kg dose group during lactation. Decreased implant numbers, mean numbers of pups born/litter and numbers of live pups born/litter was observed in the 40 mg/kg bw dose group. Pup weight was reduced on lactation days 0, 4 and 7 (males) and 4, 7 and 21 (females) in the 4.0 mg/kg bw dose group and in males at lactation day 21 (40 mg/kg bw dose group). Runting was observed in males (Lactation Day; LD 4) and female (LD 4 and 7) animals in the 4.0 mg/kg bw dose group. Reduced postnatal-day 26 pup weight was observed in male pups in the 40 mg/kg bw dose group and female pups in the 4.0 mg/kg bw dose group.