Differential MS2 Interaction with Food Contact Surfaces Determined by Atomic Force Microscopy and Virus Recovery.

Enteric viruses are recognized as major etiologies of U.S. foodborne infections. These viruses are easily transmitted via food contact surfaces. Understanding virus interactions with surfaces may facilitate the development of improved means for their removal, thus reducing transmission. Using MS2 coliphage as a virus surrogate, the strength of virus adhesion to common food processing and preparation surfaces of polyvinyl chloride (PVC) and glass was assessed by atomic force microscopy (AFM) and virus recovery assays. The interaction forces of MS2 with various surfaces were measured from adhesion peaks in force-distance curves registered using a spherical bead probe preconjugated with MS2 particles. MS2 in phosphate-buffered saline (PBS) demonstrated approximately 5 times less adhesion force to glass (0.54 nN) than to PVC (2.87 nN) (P < 0.0001). This was consistent with the virus recovery data, which showed 1.4-fold fewer virus PFU recovered from PVC than from glass after identical inoculations and 24 h of cold storage. The difference in adhesion was ascribed to both intrinsic chemical characteristics and the substrate surface porosity (smooth glass versus porous PVC). Incorporating a surfactant micellar solution of sodium dodecyl sulfate (SDS) into the PBS reduced the adhesion force for PVC (∼0 nN) and consistently increased virus recovery by 19%. With direct and indirect evidence of virus adhesion, this study illustrated a two-way assessment of virus adhesion for the initial evaluation of potential means to mitigate virus adhesion to food contact surfaces.IMPORTANCE The spread of foodborne viruses is likely associated with their adhesive nature. Virus attachment on food contact surfaces has been evaluated by quantitating virus recoveries from inoculated surfaces. This study aimed to evaluate the microenvironment in which nanometer-sized viruses interact with food contact surfaces and to compare the virus adhesion differences using AFM. The virus surrogate MS2 demonstrated less adhesion force to glass than to PVC via AFM, with the force-contributing factors including the intrinsic nature and the topography of the contact surfaces. This adhesion finding is consistent with the virus recoveries, which were determined indirectly. Greater numbers of viruses were recovered from glass than from PVC, after application at the same levels. The stronger MS2 adhesion onto PVC could be interrupted by incorporating a surfactant during the interaction between the virus and the contact surface. This study increases our understanding of the virus adhesion microenvironment and indicates ways to mitigate virus adhesion onto contact surfaces.

Growth of Salmonella during sprouting of alfalfa seeds associated with salmonellosis outbreaks.

Growth of Salmonella was assessed during sprouting of naturally contaminated alfalfa seeds associated with two outbreaks of salmonellosis. Salmonella was determined daily in sprouts and sprout rinse water samples by a three-tube most probable number (MPN) procedure and a commercial enzyme immunoassay (EIA). Growth of Salmonella in the sprouts was reflected in the rinse water, and the MPNs of the two samples were generally in agreement within approximately 1 log. The results from EIA testing of sprouts and water samples were also in agreement. The pathogen was present in the seed at less than 1 MPN/g, and it increased in number to maximum population levels of 102 to 10(3) MPN/g in one seed lot and 10(2) to 10(4) MPN/ g in the other seed lot. Maximum populations of the pathogen were apparent by day 2 of sprouting. These results show the ability of the pathogen to grow to detectable levels during the sprouting process, and they provide support for the recommendation to test the sprout water for the presence of pathogens 48 h after starting seed sprouting. The effectiveness of a 10-min, 20,0000-microg/ml (ppm) calcium hypochlorite treatment of the outbreak-associated seeds was studied. For both seed lots, the hypochlorite treatment caused a reduction, but not elimination, of Salmonella contamination in the finished sprouts. These results confirm the need to test each production batch for the presence of pathogens, even after 20,000 microg/ml (ppm) hypochlorite treatment of seeds, so that contaminated product is not distributed.

Psychological adjustment to familial genetic risk assessment: differences in two longitudinal samples.

Heritable cancer risk assessment is an increasingly common method of deriving valuable information relevant to deciding on appropriate screening regimens and preventive treatments. Assessments of heritable risk typically include familial-genetic evaluation, where analyses relate family pedigree to cancer risk, and DNA testing, where analyses indicate genetic mutations associated with cancer risk (e.g., BRCA1/BRCA2 mutations) or their absence. In this paper we report on the psychological responses of women given familial-genetic evaluations for ovarian cancer risk. The baseline and 6 to 12 follow-up assessments of an initial clinic-attending cohort of 65 women are compared with the baseline and 9 to 12 follow-up assessments of a second clinic-attending cohort of 60 women. Sizeable differences were found in the prevalence of clinically significant depression in these two physician or self-referred populations, as assessed by the Center for Epidemiological Studies Depression scale and in the mean scores. Hypotheses accounting for these differences are discussed.

A longitudinal study of psychological adjustment to familial genetic risk assessment for ovarian cancer.

OBJECTIVES: To evaluate the psychological adjustment of women during initial genetic ovarian cancer risk assessment and at clinic follow-up, 6-12 months later. METHODS: Sixty-five subjects were assessed with the Centre for Epidemiological Studies Depression Scale (CESD), Spielberger's State Anxiety Inventory, and an 18-item, investigator-designed questionnaire yielding self-report on screening responses, worry about increased risk, identification of cancer-related deaths in relatives, worry about future cancer risks of daughters, alteration of future plans as a result of ovarian cancer risk, etc. RESULTS: Thirty-three percent of subjects had CESD scores above the established cutoff for depression at baseline and 38% had scores above cutoff at follow-up. Sixteen percent of subjects had state scores on the State-Trait Anxiety Inventory higher than 1 standard deviation above average (norm) at baseline, while only 6% had scores higher than 1 SD above average at follow-up. CONCLUSION: To identify factors associated with self-reported depression at follow-up, a series of demographic and self-reported variables (e.g., presence of identified problems in family, impact of genetic risk information, concern for daughter in the future) were entered in a multiple regression analysis with the CESD follow-up score as the dependent variable. Only one predictor accounted for a significant amount of variance in depression scores. Concern for daughter's risk in the future was associated with higher depression scores at follow-up (R = 0.33, P<0.02, R(2) = 11%).

Comparison of methods for determining the presence of Escherichia coli O157:H7 in apple juice.

Six methods were compared for detection of three strains of Escherichia coli O157:H7 in enrichments of inoculated apple juice. Juice was inoculated at levels varying from 0.1 to 100 CFU/ml and centrifuged after overnight storage at 4 degrees C, and pellets were incubated at 37 degrees C in nonselective enrichment broth. At hourly intervals between 5 and 10 h and at 24 h, the enrichments were tested for E. coli O157:H7 by direct fluorescent antibody (DFA), antibody-direct epifluorescent filter technique (Ab-DEFT), direct selective plating on sorbitol MacConkey agar (SMA), immunomagnetic separation coupled to either selective plating (IMS-SMA) or the polymerase chain reaction (IMS-PCR), and flow cytometry (FC). The most consistent detection of 0.1 CFU/ml of the slowest growing strain of the pathogen was provided by the IMS-SMA and IMS-PCR after 8 h of enrichment. The time required for detection at the level of 0.1 CFU/ml for each assay was Ab-DEFT, 11 h; IMS-PCR, 16 h; FC, 24 h; IMS-SMA, 32 h; and SMA, 48 h. Absolute detection limits (without enrichment) were: IMS-PCR, 10(3) CFU/ml; Ab-DEFT and IMS-SMA, 10(4) CFU/ml; SMA, 10(5) CFU/ml; and DFA, 10(6) CFU/ml. Recovery of the pathogen (10 CFU/ml) in apple juice after 28 days of 4 degrees C storage was possible by means of an 8-h enrichment and Ab-DEFT, IMS-PCR, or IMS-SMA.

Evaluation of DNA preparation techniques for detection of the SLT-1 gene of Escherichia coli O157: H7 in bovine faeces using the polymerase chain reaction.

The polymerase chain reaction (PCR) has the potential to detect low levels of the human pathogen Escherichia coli O157: H7 in bovine faeces. To improve the utility of PCR for this application, several methods for preparing template DNA from bovine faeces, both directly and after non-selective enrichment, were tested. These were boiling, enzyme treatment, enzyme treatment plus phenol-chloroform extraction, and enzyme treatment plus phenol-chloroform extraction plus Geneclean purification. Of these, the boiling method was the most consistent and had a sensitivity of approximately 3 cfu g-1 faeces, with an assay time of less than 32 h. The boiling method was also combined with immunomagnetic separation (IMS) to detect E. coli O157: H7 in less than 8 h, but with a sensitivity of approximately 10(3) cfu g-1 faeces. These methods can be used to prepare template for PCR screening of bovine faeces using any appropriate PCR primers.

Comparison of antibody-direct epifluorescent filter technique with the most probable number procedure for rapid enumeration of Listeria in fresh vegetables.

The antibody-direct epifluorescent filter (Ab-DEFT) technique was evaluated as a rapid alternative to the most probable number (MPN) method for enumeration of artificially inoculated Listeria monocytogenes in ready-to-eat packaged salads and other fresh vegetables. Ab-DEFT was performed by homogenization of food in mesh-lined Stomacher bags, followed by prefiltration of homogenate through a 5 microns pore nylon filter, and passage of filtrate through a 0.4 micron pore black polycarbonate filter to collect and concentrate Listeria cells. After cells were stained with a fluorochrome-labeled polyclonal antibody to Listeria, the filter surface was examined by epifluorescence microscopy, and fluorescent cells were counted. A 3-tube MPN procedure was performed by successive enrichments of homogenized foods in Listeria enrichment and Fraser broths, followed by selective plating. Ab-DEFT provided quantitative determinations of Listeria cells that correlated with plate counts and MPN estimates in a linear response over a range of cell concentrations from 10 to 10(7) colony forming units (CFU)/mL. Microbial backgrounds as high as 10(8) CFU/mL did not affect performance of Ab-DEFT. In contrast to the MPN method, which required 5 days to perform, quantitation by Ab-DEFT could be completed in less than 1 h. Despite cross-reactivities demonstrated by the polyclonal fluorescent antibody, the potential of Ab-DEFT as a rapid alternative to MPN for microbial cell enumeration was evident.

Quantitative analysis and isolation of Escherichia coli O157:H7 in a food matrix using flow cytometry and cell sorting.

Flow cytometry is a potentially valuable analytical method in microbiology providing the ability to analyze rapidly large numbers of individual microorganisms by several parameters. With a flow cytometer with enhanced light scatter sensitivity and a conventionally configured sorting cytometer, a series of comparative studies to determine the ability of the two flow systems and the antibody-direct epifluorescent filter technique (Ab-DEFT) to detect and enumerate Escherichia coli O157:H7 were made. Initial experiments used culture-derived mixtures of non-pathogenic E. coli and serial dilutions of E. coli O157:H7. Subsequent studies involved analysis of enrichment cultures from ground beef inoculated with E. coli O157:H7. Comparison of flow cytometry with microscopy and plate counts produced similar results at higher concentrations in both culture mixtures and beef enrichments. At the lowest concentrations Ab-DEFT was more sensitive, however, the time required for analysis was much less with flow cytometry. With a cytometer with enhanced light scatter sensitivity designed for bacterial analysis, O157:H7 could be distinguished from E. coli strain HB101 on the basis of light scatter. This instrument also provided direct count data for selected populations. In experiments using cell sorting to isolate target organisms, the purity of fluorescent-labeled E. coli O157:H7 sorted from beef enrichment cultures and plated was not affected by the level of background organisms, as is often the case in conventional plating procedures.

Rapid identification of Escherichia coli O157:H7 in bovine feces using the antibody-direct epifluorescent filter technique (Ab-DEFT).

The antibody-direct epifluorescent filter technique (Ab-DEFT) was adapted for direct detection of Escherichia coli O157:H7 in bovine feces. The method involved suspension of bovine feces in buffer, centrifugation for 30 s, treatment of the supernatant with trypsin and Triton X-100 at 50 degrees C for 10 min, pre-filtration through 5 and 1.2 microns pore filters, and filtration through a 0.4 micron pore filter. The final filter was stained with fluorescein-labeled polyclonal antibody specific for the O157 antigen and examined by epifluorescence microscopy. The Ab-DEFT was correlated with viable plate counts for enumeration of the pathogen in artificially inoculated bovine feces (r = 0.96). The limit of detection was approximately 10(4)-10(5) CFU/g feces. The procedure provided a clean background for microscopic visualization of cells; however, cell loss and inaccurate quantitation sometimes resulted. E. coli O157:H7 was detected in feces of an inoculated calf for more than 3 weeks post-inoculation. The Ab-DEFT may be useful for rapid screening of cattle for the presence of the E. coli O157:H7 and as an analytical method in ecological studies of the pathogen.

Relationship of perceived barriers to breast self-examination in women of varying ages and levels of education.

PURPOSE/OBJECTIVES: To examine perceived barriers to performing breast self-examination (BSE) in women of varying age groups and education levels and investigate the relationship of age and education to frequency of BSE. DESIGN: Descriptive, retrospective. SETTING: A university nursing center and clerical offices. SAMPLE: Convenience sample of 374 women. METHOD: Subjects were asked to complete Champion's Health Belief Model questionnaire. MAIN RESEARCH VARIABLE: Barriers to BSE practice. FINDINGS: Chi-squares analyses comparing age and education levels to BSE frequency showed no significant differences among groups. Cross tabulations suggested that differences in perception of individual barriers to practice may exist among women of different age groups and education levels. CONCLUSION: Women of varying age groups and education levels responded to individual barrier items differently. IMPLICATIONS FOR NURSING PRACTICE: Young women may need help developing confidence in their BSE technique as well as accurate information to reduce their fears about BSE. Middle-aged women also need this information to reduce their fears and may need help in developing a reminder method for practicing BSE. Older women may benefit from education about BSE's value to them. Less educated women may need information to reduce fear, while women at higher education levels may benefit from help in developing a reminder method.

Antibody-direct epifluorescent filter technique for rapid, direct enumeration of Escherichia coli O157:H7 in beef.

Artificially inoculated Escherichia coli O157:H7 was directly enumerated in ground beef and beef exudate, without enrichment or selection, by the antibody-direct epifluorescent filter technique (Ab-DEFT). The total assay time of the Ab-DEFT was less than 1 h. The beef was homogenized, treated for 15 min with trypsin and Triton X-100, and passed through a 5-microns-pore-size prefilter and then through a 0.2-microns-pore-size black polycarbonate filter. The final filter was stained directly with fluorescein-labeled anti-O157 polyclonal antibody, rinsed, and examined by epifluorescence microscopy. The sensitivity of the Ab-DEFT was compared with that of a standard enrichment culture technique. Both methods reliably determined the presence of the pathogen in beef at 16 CFU/g. The Ab-DEFT was also useful for quantifying the pathogen and monitoring its growth in beef.