Short communication: Microbiological quality of raw cow milk and its association with herd management practices in Northern China.

Contamination of raw milk with bacterial pathogens is potentially hazardous to human health. The aim of this study was to evaluate the total bacteria count (TBC) and presence of pathogens in raw milk in Northern China along with the associated herd management practices. A total of 160 raw milk samples were collected from 80 dairy herds in Northern China. All raw milk samples were analyzed for TBC and pathogens by culturing. The results showed that the number of raw milk samples with TBC <2 × 106 cfu/mL and <1 × 105 cfu/mL was 146 (91.25%) and 70 (43.75%), respectively. A total of 84 (52.50%) raw milk samples were Staphylococcus aureus positive, 72 (45.00%) were Escherichia coli positive, 2 (1.25%) were Salmonella positive, 2 (1.25%) were Listeria monocytogenes positive, and 3 (1.88%) were Campylobacter positive. The prevalence of S. aureus was influenced by season, herd size, milking frequency, disinfection frequency, and use of a Dairy Herd Improvement program. The TBC was influenced by season and milk frequency. The correlation between TBC and prevalence of S. aureus or E. coli is significant. The effect size statistical analysis showed that season and herd (but not Dairy Herd Improvement, herd size, milking frequency, disinfection frequency, and area) were the most important factors affecting TBC in raw milk. In conclusion, the presence of bacteria in raw milk was associated with season and herd management practices, and further comprehensive study will be powerful for effectively characterizing various factors affecting milk microbial quality in bulk tanks in China.

Microbial degradation of gasoline in soil: Effect of season of sampling.

In cases where fire debris contains soil, microorganisms can rapidly and irreversibly alter the chemical composition of any ignitable liquid residue that may be present. In this study, differences in microbial degradation due to the season in which the sample is collected was examined. Soil samples were collected from the same site during Fall, Winter, Spring and Summer and the degradation of gasoline was monitored over 30 days. Predominant viable bacterial populations enumerated using real-time PCR and reverse transcriptase polymerase chain reaction (RT-PCR) enumeration revealed the predominant viable bacterial genera to be Alcaligenes, Bacillus, and Flavobacterium. Overall, the compounds most vulnerable to microbial degradation are the n-alkanes, followed by the mono-substituted alkylbenzenes (e.g., toluene, ethylbenzene, propylbenzene and isopropylbenzene). Benzaldehyde (a degradation product of toluene) was also identified as a marker for the extent of biodegradation. Ultimately, it was determined that soil collected during an unusually hot and dry summer exhibited the least degradation with little to no change in gasoline for up to 4 days, readily detectable n-alkanes for up to 7 days and relatively high levels of resilient compounds such as o-xylene, p-xylene and 1,3,5-trimethylbenzene. These results demonstrate, however, that prompt preservation and/or analysis of soil evidence is required in order to properly classify an ignitable liquid residue.

A multicentre randomized trial of the treatment of patients with pemphigus vulgaris with infliximab and prednisone compared with prednisone alone.

BACKGROUND: Pemphigus vulgaris (PV) is a blistering disease and tumour necrosis factor-α has a role in its pathogenesis. OBJECTIVES: To evaluate the safety of infliximab (IFX) with prednisone compared with prednisone alone in the treatment of PV. In addition, treatment response was assessed and mechanistic studies were performed. METHODS: Subjects with PV who had ongoing disease activity while being maintained on prednisone were randomized to receive either IFX or placebo in addition to prednisone. Response status and immunoglobulin (Ig) G anti-desmoglein (Dsg)1 and Dsg3 antibodies were assessed at 18 and 26 weeks. RESULTS: Ten subjects were randomized to each group. There were no safety signals during the course of the study. At week 18, one subject in each group had responded. At week 26, three IFX-treated subjects vs. none in the placebo group had responded (P = 0·21). At weeks 18 and 26, the median IgG anti-Dsg1 and anti-Dsg3 levels were lower in the IFX-treated patients [IgG anti-Dsg-1 (week 18, P = 0·035; week 26, P = 0·022); IgG anti-Dsg3 (week 18, P = 0·035; week, 26 P = 0·05)]. CONCLUSIONS: This study is limited by the relatively small sample size. There was no significant difference between study arms in the proportion of subjects with treatment-related adverse events > grade 3. IFX therapy was not shown to be effective for the treatment of patients with PV in this randomized, placebo-controlled trial, although IFX treatment may be associated with a decrease in anti-Dsg1 and Dsg3 antibodies.

Detection and recovery of sublethally-injured enterotoxigenic Staphylococcus aureus.

AIMS: To determine whether sublethally-injured (acid- or heat-shocked) Staphylococcus aureus cells are recoverable using selective agar overlays. METHODS AND RESULTS: Brain Heart Infusion (BHI) Agar overlaid with either Baird-Parker Agar (BPA) or Gram-Positive Agar (GPA) was compared in the ability to resuscitate heat- and acid-shocked enterotoxigenic Staph. aureus. BHI/BPA overlays allowed for greater recovery of both heat- and acid-shocked cells than BHI/GPA, although the former was not selective and allowed growth of bacteria other than Staph. aureus. No significant difference existed in percent recovery of heat- and acid-shocked cells between the two overlay approaches. Significant differences were noted in counts on BHI/GPA plates and straight selective GPA/GPA plates, however. Viability of heat- and acid-shocked Staph. aureus was also examined using fluorescence microscopy, the relative counts of which correlated well to the calculated percent recovery on selective agar overlays. CONCLUSIONS: This work has shown that an improved agar overlay technique increases the sensitivity of the standard plate count while enumerating sublethally-injured enterotoxigenic Staph. aureus compared with direct plating onto selective media. SIGNIFICANCE AND IMPACT OF THE STUDY: These data emphasize the need to develop practical and cost-effective methods that reliably detect and enumerate sublethally-injured pathogens such as Staph. aureus.

Development of a multiplex polymerase chain reaction assay for detection and differentiation of Staphylococcus aureus in dairy products.

A multiplex polymerase chain reaction (PCR) assay was developed for the detection and differentiation of enterotoxigenic Staphylococcus aureus in dairy products. A solvent extraction procedure was successfully modified for extraction of S. aureus DNA from 10 ml of artificially contaminated skim milk or 20 g cheddar cheese. Primers targeting the enterotoxin C gene (entC) and thermostable nuclease gene (nuc) were used in the multiplex PCR. PCR products were confirmed using restriction fragment length polymorphism analysis. DNA was consistently quantified and amplified by uniplex PCR from 10 CFU/ml of S. aureus in skim milk or 10 CFU/20 g cheddar cheese. The sensitivity of the multiplex PCR was 100 CFU/ml of skim milk or 100 CFU/20 g cheddar cheese. The developed methodology allows presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h.

Prevalence and expression of enterotoxins in Bacillus cereus and other Bacillus spp., a literature review.

Members of the Bacillus genus are ubiquitous soil microorganisms and are generally considered harmless contaminants. However, a few species are known toxin producers, including the foodborne pathogen, B. cereus. This species produces two distinct types of foodborne illness, the emetic (vomit-inducing) syndrome, associated with consumption of toxin in cooked rice dishes, and the diarrheal illness seen occasionally following consumption of contaminated meats, sauces, and certain dairy products. In the latter case, illness results from the production of enterotoxins by vegetative cells in the small intestine of the host. In dairy products, the occurrence of Bacillus spp. is inevitable, and the spore-forming ability of this organism allows it to easily survive pasteurization. Many strains have been shown to grow and produce enterotoxin in dairy products at refrigeration temperatures. Evaluation of toxin gene presence and toxin expression in Bacillus spp. other than B. cereus has not been thoroughly investigated. However, the presence of natural isolates of Bacillus spp. harboring one or more enterotoxin gene(s) and subsequent demonstration of conditions which may support toxin expression holds crucial importance in the food safety arena.

A comparison of methods for the detection of Escherichia coli O157:H7 from artificially-contaminated dairy products using PCR.

Rapid nucleic acid-based methods to detect human pathogens in foods are dependent on the reliability of the DNA or RNA extraction method used. Skim milk, non-fat dry milk, Cheddar and Brie cheese, and reconstituted whey powder were seeded with serially diluted (10(0)-10(7) cfu 10 ml(-1)) Escherichia coli O157:H7 and subjected to DNA extraction (i) directly from the food product using a solvent-based procedure and (ii) using a guanidinium isothiocyanate (GITC) procedure after previous bacterial concentration. Both the efficiency of DNA extraction and the overall PCR detection limits were evaluated. In almost all instances, the total DNA yield using the solvent method was greater than that obtained for the concentration method. However, the purity of the DNA obtained after bacterial concentration was significantly better than that obtained after organic extraction alone. PCR detection limits after each DNA recovery method varied with the specific food, ranging from 10(1) to 10(4) cfu ml(-1) for all products except whey powder. DNA yields and subsequent PCR detection limits for reconstituted whey powder were extremely poor, and neither procedural changes nor the addition of PCR enhancement agents were able to improve recovery and/or detection. It is concluded that the efficiency of DNA extraction is an extremely important and frequently overlooked variable impacting the overall detection limits of PCR-based detection strategies.

Molecular beacon polymerase chain reaction detection of Escherichia coli O157:H7 in milk.

A fluorescently labeled oligonucleotide probe (molecular beacon) was applied to detect Escherichia coli O157:H7 in artificially contaminated skim milk during polymerase chain reaction (PCR) amplification of extracted DNA. The probe was designed to hybridize with a region of the slt-II gene coding for the A subunit and to fluoresce when the hairpin-stem conformation was linearized upon hybridization to the target sequence. The molecular beacon was incorporated into PCR reactions containing DNA extracted from artificially contaminated skim milk. The degree of fluorescence was monitored in PCR reactions containing 10(3), 10(5), and 10(7) CFU of E. coli O157:H7 per ml and was found to correlate with the amount of template in each reaction. Fluorescence significantly increased above background levels by cycle 8, 14, or 14 in reactions containing DNA from the 10(7)-, 10(5)-, or 10(3)-CFU/ml template, respectively (P < 0.05). Molecular beacon PCR demonstrated positive results more rapidly than traditional agarose gel electrophoresis analysis of PCR products. Use of molecular beacons allows real-time monitoring of PCR reactions, and the closed-tube format allows simultaneous detection and confirmation of target amplicons without the need for agarose gel electrophoresis and/or Southern blotting. This is the first report of a stem-and-loop molecular beacon being applied for direct detection of a pathogen in food.

Survival of Escherichia coli O157:H7 in buttermilk as affected by contamination point and storage temperature.

The effects of contamination point (during fermentation versus postfermentation) and storage temperature (5 and 12 degrees C) were determined for survival of Escherichia coli O157:H7 in fermented buttermilk. E. coli O157:H7 was recovered from buttermilk inoculated during fermentation for 22 days and in buttermilk inoculated postfermentation for 32 days. For storage temperatures of 5 and 12 degrees C, D-values were lower for E. coli O157:H7 inoculated during fermentation (2.5, 2.2 days) than postfermentation (5.6, 4.8 days) (P < 0.05). Developed acidity in inoculated buttermilks was not different from controls (P > 0.05). The extended recovery of viable enterohemorrhagic E. coli O157:H7 from both processing scenarios indicates that the presence of E. coli O157:H7 in buttermilk is not limited to postprocessing contamination.

Nucleic acid persistence in heat-killed Escherichia coli O157:H7 from contaminated skim milk.

Polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR using primers targeting 16S rRNA sequences in Escherichia coli O157:H7 were applied to monitor the stability of rDNA and rRNA in cells killed by mild heat treatment (60 degrees C) in skim milk. Serial dilutions of purified RNA and DNA from E. coli O157:H7 in skim milk were amplified by RT-PCR or PCR, respectively, before heat treatment and at time points 0, 6, 12, 24, and 48 h after heating. In general, DNA-PCR provided stronger amplification signals compared to RT-PCR at the corresponding time points with the same PCR primer set, indicating a lower efficiency of RNA amplification compared to that of DNA. Ribosomal RNA and rDNA could be amplified by RT-PCR or PCR from both viable and dead cells throughout the 48-h posttreatment holding period. For RT-PCR, amplification signals decreased in intensity with increased holding time, while the efficiency of amplification of DNA sequences from dead cells remained fairly stable throughout the study. DNA persistence was greater than that of rRNA following cell death by mild heat treatment in skim milk. Skim milk did not appear to accelerate nucleic acid degradation. While rRNA was less stable than DNA, its detection by RT-PCR may not be appropriate as an exclusive indicator of cell viability in minimally processed foods.

rRNA stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7.

Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens, Escherichia coli O157:H7 and enterotoxigenic Staphylococcus aureus, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37 degrees C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1, 400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121 degrees C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80 degrees C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells.

Understanding the three national databases on collegiate alcohol and drug use.

An overview of the three major databases used to examine alcohol and other drug use habits of American college students is provided. The databases are compared in terms of purpose, study population, subject selection, method of administration, focus, utility for institutional use, and trend analyses. The authors conclude that no one source of data is "best." Rather, the studies represent three different sources of data. Although information from these databases overlaps to some extent, each database makes a unique contribution to the field.

Biochemical characterization, developmental expression, and induction of the immune protein scolexin from Manduca sexta.

The immune protein, scolexin, a bacteria-induced, larva-specific protein from Manduca sexta, was shown to exist in the hemolymph in two isoelectric forms designated herein as scolexin-1 and scolexin-2 (native M(r) approximately 72 kd). These two charge isomers appeared to share the same amino acid composition. Scolexin is composed of two subunits (peptide M(r) approximately 36 kd) that possess the same N-terminus. Scolexin-2 was subjected to glycosyl composition analysis, revealing the presence of galactose, glucose, mannose, xylose, and sialic acid residues. Hybridization of epidermal RNA with oligonucleotides deduced from the scolexin N-terminal sequence showed a continuous decline in mRNA following day 0 of the 5th larval instar. By employing in vitro protein labelling, it was found that organ cultures of the epidermis from immune larvae showed a greater ability over that of naive epidermal cultures to synthesize scolexin; these data reflected the inducible response seen in the hemolymph, and confirm other data indicating that the epidermis is an important site of scolexin biosynthesis.

Measuring patient perceptions of the patient-doctor interaction. Development of the PDIS.

Patient satisfaction has been related to patient compliance, doctor-patient information exchange, and continuity of care. However, the construct of patient satisfaction is not well defined; its measurement has typically consisted of the anecdotal generation of questions that may not tap issues of concern to patients. Generally, neither reliability nor validity of the measurement instruments are considered. The present study investigated the construct of patient satisfaction and developed the Smith-Falvo Patient-Doctor Interaction Scale (PDIS), a patient satisfaction scale based on empirically derived dimensions of patient satisfaction. The project consisted of three studies. The first study involved the generation of physician behavior descriptions by 22 family practice center patients. The resultant 1540 descriptions were grouped into 80 reliable categories. In Study 2, a sample of 30 patients rated the behavior descriptions on a 9-point scale from most preferred to least preferred. Multidimensional scaling techniques were applied to the preference ratings, resulting in the identification of two dimensions: general health care delivery and inappropriate interpersonal interactions. The PDIS was then constructed from items salient to these two dimensions. Outcome of the investigation of the reliability and validity of the PDIS in a third study was quite positive.

Comparison of cardiovascular response to combined static-dynamic effort, postprandial dynamic effort and dynamic effort alone in patients with chronic ischemic heart disease.

The cardiovascular responses to combined static-dynamic effort, postprandial dynamic effort and dynamic effort alone were evaluated by upright bicycle ergometry during equilibrium-gated blood pool scintigraphy in 24 men, mean age 59 +/- 8 years, with chronic ischemic heart disease. Combined static-dynamic effort and the postprandial state elicited a peak cardiovascular response similar to that of dynamic effort alone; work load 643 +/- 156 and 638 +/- 161 vs 650 +/- 153 kg-m/min, respectively; heart rate 147 +/- 14 and 145 +/- 14 vs 143 +/- 17 beats/min; systolic pressure 195 +/- 26 and 200 +/- 25 vs 197 +/- 25 mm Hg; and rate-pressure product 286 +/- 48 and 292 +/- 55 vs 282 +/- 52. Heart rate, intraarterial systolic and diastolic pressures, rate-pressure product and ejection fraction were similar for the three test conditions at the onset of ischemia and at peak effort. The prevalence and extent of exercise-induced ischemic left ventricular dysfunction, ST-segment depression, angina pectoris and ventricular ectopic activity were also similar during the three test conditions. Direct and indirect measurements of systolic and diastolic blood pressure were highly correlated. The onset of ischemic ST-segment depression and angina pectoris correlated as strongly with heart rate alone as with the rate-pressure product during all three test conditions. The cardiovascular response to combined static-dynamic effort and to postprandial dynamic effort becomes more similar to that of dynamic effort alone as dynamic effort reaches a symptom limit. If significant ischemic and arrhythmic abnormalities are absent during symptom-limited dynamic exercise testing, they are unlikely to appear during combined static-dynamic or postprandial dynamic effort. This simplifies, the task of formulating guidelines for physical effort in patients with chronic ischemic heart disease, especially in providing "clearance" to perform avocational and vocational tasks involving combined static-dynamic and postprandial dynamic effort.

Program evaluation in sex education: outcome assessment of sexual awareness weekend workshops.

The results of an outcome assessment of a growth-oriented sex education program are reported. The evaluation project attempted to overcome flaws of existing sex education research by explicitly delineating goals and by incorporating both a rigorous experimental control group design and quasiexperimental cohort comparisons. A Sex Questionnaire (covering sexual knowledge, attitudes, and behavior) was administered to the participants of three weekend workshops at the tiem of registration and 1 month later. Overflow resistration for one workshop permitted the random selection of a no-program control group. Results supported inferences of workshop effectiveness in meeting program goals in the areas of attitude and behavior change, but not with regard to increasing sexual knowledge. Implications for development and evaluation of sex education programs are discussed.

PIP: Several voluntary, growth-oriented, intensive sex education workshops were given for students at the Southern Illinois University campus. To facilitate evaluation of the program's effectiveness, goals were defined beforehand and a rigorous experimental control group design and quasiexperimental cohort comparison were built into the program. A sex questionnaire, covering sexual attitudes, knowledge, and behavior, was administered before and after the course. All data are tabulated. Results of the comparisons show that attitudes and behavior were changed significantly by the workshops. Participants gained more knowledge regarding sex than nonparticipants, but the difference was not significant. Attitudes became more tolerant and the incidence of masturbation, the only type of sex behavior possible without a partner, increased. The higher the level of the subject's father's education, the more likely that the workshops would effect changes. In fact, participants with a high social status, religious liberalness, and a prior level of sexual knowledge, were the most likely to change as a result of the workshops. All of these predictor variables were more strongly related to behavioral than to attitudinal changes. The study is seen to have strong external validity for its conclusions.

Impact evaluation of service programs: three flexible designs.

PIP: Flexibility in evaluative research does not necessitate the abandonment of randomly constructed comparison groups. 3 designs (cohort control, no preference, and intensive or minimal program) are reviewed which provide at least the option of randomization, while maintaining great flexibility. The strengths and weaknesses of each are discussed. It is noted that, as evaluation research is undertaken to supply information to aid decision making, the clarity of the information is of great importance. Randomly selected comparison groups enhance the interpretability of findings. The 3 designs presented provide flexibility in the use of comparison groups, recognizing and making trade-offs necessitated by political or logistical factors. In almost all cases, program administrators will find evidence of program effectiveness useful.^ieng