Molecular ecology of Listeria spp., Salmonella, Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli in pristine natural environments in Northern Colorado.

AIMS: Molecular subtyping is commonly used in foodborne disease surveillance and microbial source tracking. There is a knowledge gap regarding the molecular ecology of foodborne pathogens in non-food-associated environments. The objective of this study was to isolate and subtype foodborne pathogens from pristine natural environments with minimal anthropogenic inputs. MATERIALS AND RESULTS: Five locations (wilderness areas) in Northern Colorado were sampled during the spring, summer and fall over a 2-year period. Soil, water, sediment, surface soil and wildlife faecal samples were microbiologically analysed to detect Listeria, Salmonella and Shiga toxin-producing Escherichia coli (STEC), and resultant isolates were subtyped. Three samples tested positive for Listeria monocytogenes and 19 samples contained other Listeria spp. Salmonella was isolated from two samples, five samples contained non-O157 STEC, and E. coli O157:H7 was not detected. Two L. monocytogenes isolates from faecal samples collected from the same wilderness area over a year apart shared the same PFGE pattern, while all other isolates had a unique type. CONCLUSIONS: Our data indicate that (i) there was a rare presence of human foodborne pathogens in pristine natural environments in Northern Colorado, (ii) there was genetic diversity between organisms isolated within a given wilderness area, and (iii) the Northern Colorado climate and topography may contribute to the low occurrence of these organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Relatively little is known about the molecular ecology of foodborne pathogens in pristine natural environments. While foodborne pathogens were rarely detected in wildlife faecal and environmental samples from the wilderness areas in this study, some isolates shared DNA fingerprint types with human clinical isolates from same region during the same time frame, highlighting the need for environmental isolate subtype data. The availability of molecular subtyping data for non-food-associated foodborne pathogen isolates can facilitate epidemiological and microbial source tracking investigations.

Sources of Klebsiella and Raoultella species on dairy farms: be careful where you walk.

Klebsiella spp. are a common cause of mastitis, milk loss, and culling on dairy farms. Control of Klebsiella mastitis is largely based on prevention of exposure of the udder to the pathogen. To identify critical control points for mastitis prevention, potential Klebsiella sources and transmission cycles in the farm environment were investigated, including oro-fecal transmission, transmission via the indoor environment, and transmission via the outdoor environment. A total of 305 samples was collected from 3 dairy farms in upstate New York in the summer of 2007, and included soil, feed crops, feed, water, rumen content, feces, bedding, and manure from alleyways and holding pens. Klebsiella spp. were detected in 100% of rumen samples, 89% of water samples, and approximately 64% of soil, feces, bedding, alleyway, and holding pen samples. Detection of Klebsiella spp. in feed crops and feed was less common. Genotypic identification of species using rpoB sequence data showed that Klebsiella pneumoniae was the most common species in rumen content, feces, and alleyways, whereas Klebsiella oxytoca, Klebsiella variicola, and Raoultella planticola were the most frequent species among isolates from soil and feed crops. Random amplified polymorphic DNA-based strain typing showed heterogeneity of Klebsiella spp. in rumen content and feces, with a median of 4 strains per 5 isolates. Observational and bacteriological data support the existence of an oro-fecal transmission cycle, which is primarily maintained through direct contact with fecal contamination or through ingestion of contaminated drinking water. Fecal shedding of Klebsiella spp. contributes to pathogen loads in the environment, including bedding, alleyways, and holding pens. Hygiene of alleyways and holding pens is an important component of Klebsiella control on dairy farms.

A method for accurate localization of the first heart sound and possible applications.

We have previously developed a method for localization of the first heart sound (S1) using wavelet denoising and ECG-gated peak-picking. In this study, an additional enhancement step based on cross-correlation and ECG-gated ensemble averaging (EA) is presented. The main objective of the improved method was to localize S1 with very high temporal accuracy in (pseudo-) real time. The performance of S1 detection and localization, with and without EA enhancement, was evaluated on simulated as well as experimental data. The simulation study showed that EA enhancement reduced the localization error considerably and that S1 could be accurately localized at much lower signal-to-noise ratios. The experimental data were taken from ten healthy subjects at rest and during invoked hyper- and hypotension. For this material, the number of correct S1 detections increased from 91% to 98% when using EA enhancement. Improved performance was also demonstrated when EA enhancement was used for continuous tracking of blood pressure changes and for respiration monitoring via the electromechanical activation time. These are two typical applications where accurate localization of S1 is essential for the results.

Cleanliness scores as indicator of Klebsiella exposure in dairy cows.

This study was designed to explore the relationship between cow and udder cleanliness scores and the risk of isolation of Klebsiella spp. from lower hind legs and teat ends, respectively. The distribution of Klebsiella species was compared among isolates from teat ends, legs, and cases of clinical mastitis obtained from 2 dairy farms in New York State, with 850 and 1,000 cows, respectively. Farms were visited twice approximately 4 wk apart in August and September 2007 to obtain cleanliness scores and swabs from legs and teats. Isolates of Klebsiella clinical mastitis from each farm were collected from July through October 2007. Two studies were conducted. In the first study, whole-cow cleanliness of a purposive sample of 200 lactating cows was scored using a 4-point scale, and swabs were taken from their lower hind legs. In the second study, udder cleanliness of a separate convenience sample of 199 lactating cows was scored in the milking parlor, and swabs were taken from their teat ends before and after premilking udder preparation. Prevalence of Klebsiella spp. on legs and teat ends before udder preparation was 59 and 60%, respectively. Logistic regression was used to explore the association between isolation of Klebsiella spp. and cleanliness scores. Cow cleanliness scores and udder cleanliness scores were not associated with detection of Klebsiella on legs and on teats before udder preparation, respectively. After udder preparation, 43% of previously Klebsiella positive teat end samples remained positive, with significant differences between farms and months. Teats from dirty udders were significantly more likely to test positive for Klebsiella after udder preparation than teats from clean udders. The proportion of Klebsiella pneumoniae and Klebsiella oxytoca isolates was similar for isolates from teat end swabs and clinical mastitis cases, supporting the notion that the presence of Klebsiella on teat ends may lead to opportunistic intramammary infections. Udder cleanliness scores could be used as a management tool to monitor the risk of exposure to Klebsiella spp. on teat ends.

Creating a framework for the prioritization of biosecurity risks to the New Zealand dairy industry.

The New Zealand dairy sector relies on robust biosecurity measures to control and mitigate a wide range of threats to the industry. To optimize the prioritization of organisms and manage the risk they pose to the sector in a transparent and credible way, the Dairy Biosecurity Risk Evaluation Framework (D-BRiEF) was developed. This comprehensive framework was specifically designed for decision support, using a standardized approach to address the full spectrum of biosecurity threats to the sector, including exotic and endemic animal disease organisms, pest plants and insects. D-BRiEF is underpinned by three main processes, namely (i) hazard identification; (ii) multicriteria risk assessment; and (iii) communication for risk management. Expert knowledge and empirical data, including associated uncertainty, are harnessed in a standardized format. Results feed into a probability-impact model that was developed in close collaboration with dairy sector economists to provide overall comparative 10-year quantitative economic impact estimates for each assessed risk organism. A description of the overarching framework, which applies to diverse organism groups, is presented with detailed methodology on both endemic and exotic animal disease risk organisms. Examples of visual outputs are included, although actual ranking results are not reported due to industry confidentiality. D-BRiEF can provide a decision advantage to DairyNZ biosecurity risk managers and sector stakeholders by creating a transparent process that can be interrogated and updated at multiple levels to fully understand the layers of risk posed by different organisms.

Inferring source attribution from a multiyear multisource data set of Salmonella in Minnesota.

Salmonella enterica is a global health concern because of its widespread association with foodborne illness. Bayesian models have been developed to attribute the burden of human salmonellosis to specific sources with the ultimate objective of prioritizing intervention strategies. Important considerations of source attribution models include the evaluation of the quality of input data, assessment of whether attribution results logically reflect the data trends and identification of patterns within the data that might explain the detailed contribution of different sources to the disease burden. Here, more than 12,000 non-typhoidal Salmonella isolates from human, bovine, porcine, chicken and turkey sources that originated in Minnesota were analysed. A modified Bayesian source attribution model (available in a dedicated R package), accounting for non-sampled sources of infection, attributed 4,672 human cases to sources assessed here. Most (60%) cases were attributed to chicken, although there was a spike in cases attributed to a non-sampled source in the second half of the study period. Molecular epidemiological analysis methods were used to supplement risk modelling, and a visual attribution application was developed to facilitate data exploration and comprehension of the large multiyear data set assessed here. A large amount of within-source diversity and low similarity between sources was observed, and visual exploration of data provided clues into variations driving the attribution modelling results. Results from this pillared approach provided first attribution estimates for Salmonella in Minnesota and offer an understanding of current data gaps as well as key pathogen population features, such as serotype frequency, similarity and diversity across the sources. Results here will be used to inform policy and management strategies ultimately intended to prevent and control Salmonella infection in the state.

Fecal shedding of Klebsiella pneumoniae by dairy cows.

Klebsiella pneumoniae is a common cause of clinical mastitis in dairy cattle. Wood products are considered to be the main source of Klebsiella on dairy farms. Environmental hygiene and use of inorganic bedding materials such as sand are recommended to control Klebsiella mastitis. However, Klebsiella mastitis still occurs on well-managed dairy farms that use sand as bedding material. In a 5-mo study in a New York State dairy herd performed during the summer of 2005, all of 9 samples of unused sand bedding tested negative for Klebsiella, whereas 14 of 18 samples of used sand bedding contained Klebsiella at a median level of 10(4.6) cfu/g. We hypothesized that fecal shedding of Klebsiella by dairy cows contributes to the presence of Klebsiella in the environment. Using a cheap and simple method based on ampicillin-containing MacConkey agar for screening, and biochemical tests for confirmation of species identity, 595 fecal samples from healthy dairy cattle were screened for presence of Klebsiella. In a longitudinal study of 100 cows followed over 5 mo, more than 80% of fecal samples tested positive for K. pneumoniae. The average prevalence of K. pneumoniae-positive fecal samples was also above 80% in a cross-sectional study of 100 cows from 10 herds across New York and Massachusetts. Fecal shedding of K. pneumoniae by a large proportion of dairy cows may explain why Klebsiella mastitis occurs in herds that use inorganic bedding material or other bedding material that is free from Klebsiella upon introduction into the barn.

Pulse wave transit time for monitoring respiration rate.

In this study, we investigate the beat-to-beat respiratory fluctuations in pulse wave transit time (PTT) and its subcomponents, the cardiac pre-ejection period (PEP) and the vessel transit time (VTT) in ten healthy subjects. The three transit times were found to fluctuate in pace with respiration. When applying a simple breath detecting algorithm, 88% of the breaths seen in a respiration air-flow reference could be detected correctly in PTT. Corresponding numbers for PEP and VTT were 76 and 81%, respectively. The performance during hypo- and hypertension was investigated by invoking blood pressure changes. In these situations, the error rates in breath detection were significantly higher. PTT can be derived from signals already present in most standard monitoring set-ups. The transit time technology thus has prospects to become an interesting alternative for respiration rate monitoring.

Procoagulant albumin increases vascular endothelial cell prostacyclin secretion.

Vascular endothelium regulates multiple aspects of platelet function through secretion of a variety of substances, including von Willebrand factor, nitric oxide, and prostacyclin (PGI2). The objective of this study was to determine whether procoagulant albumin (P-A1), a modified form of albumin present in normal human plasma could modulate endothelial cell secretion of these substances. P-A1 did not affect constitutive secretion of von Willebrand factor or nitric oxide, but did increase PGI2 secretion in a time- and concentration-dependent manner. Pre-treatment of endothelial cells with aspirin, or use of suramin, a broad-specificity inhibitor, prevented the response to P-A1. Prostaglandin H synthase-2 contributed to the P-A1-induced PGI2 secretion. These results indicate that in addition to inducing tissue factor activity and reducing protein C activation and fibrinolysis, P-A1 also modulates vascular endothelial cell PGI2 secretion, and potentially, platelet function.

Contrast sensitivity in macular degeneration and the relation to subjective visual impairment.

Contrast sensitivity was determined with electronic and computer equipment (Cadwell CTS 5000) and with optotype test charts (LH low contrast test) in 31 patients, aged 47-89, with macular degeneration, mostly of the age-related type. Values of maximal sensitivity and mean sensitivity in different spatial frequency bands were estimated. Visual acuity at distance and the magnification factor needed for reading were also determined. Each of these measures were correlated with a score obtained in a subjective evaluation of difficulties with visual discrimination and orientation. It was found that poor visual discrimination and orientation was better correlated with low contrast sensitivity than with low visual acuity. The outcome of the LH-test was a better predictor of orientation problems than the electronic test.

A respiration monitor based on electrocardiographic and photoplethysmographic sensor fusion.

Respiratory variations are present in the pulse wave transit time (PTT) and, as a consequence of respiratory sinus arrhythmia, in the electrocardiogram (ECG) and the photoplethysmogram (PPG). The aim of this study was to investigate these variations in healthy subjects during rest and invoked blood pressure changes. The primary goal was to develop a non-invasive respiration monitor. The error rates for breath detection during rest were 14%, 11% and 10% for PTT, ECG and PPG respectively. Significantly higher error rates were found in hypotension and hypertension. To improve accuracy and robustness, the signals were merged in a neural network resulting in an error rate of 9%.

Application of the Articulation Index and the Speech Transmission Index to the recognition of speech by normal-hearing and hearing-impaired listeners.

The present article is divided into four major sections dealing with the application of acoustical indices to the prediction of speech recognition performance. In the first section, two acoustical indices, the Articulation Index (AI) and the Speech Transmission Index (STI), are described. In the next section, the effectiveness of the AI and the STI in describing the performance of normal-hearing and hearing-impaired subjects listening to spectrally distorted (filtered) and temporarily distorted (reverberant) speech is examined retrospectively. In the third section, the results of a prospective investigation that examined the recognition of nonsense syllables under conditions of babble competition, filtering and reverberation are described. Finally, in the fourth section, the ability of the acoustical indices to describe the performance of 10 hearing-impaired listeners, 5 listening in quiet and 5 in babble, is examined. It is concluded that both the AI and the STI have significant shortcomings. A hybrid index, designated mSTI, which takes the best features from each procedure, is described and demonstrated to be the best alternative presently available.

Reference threshold sound-pressure levels for the TDH-50 and ER-3A earphones.

Reference threshold sound-pressure levels were established for a new insert earphone, the ER-3A tubephone, and for the TDH-50 earphone. In test-retest comparisons, the tubephone produced estimates of auditory threshold as reliable as the thresholds produced by the supraaural earphone. Reference thresholds were developed for the two earphones from data contributed by three laboratories. While the TDH-50 data are in good agreement with the provisional ANSI 6-cc coupler reference levels (ASHA, 1982), the ER-3A data are at variance with the manufacturer's provisional recommendation for 2-cc coupler reference thresholds for frequencies below 1 kHz. The differences are attributed to physiologic noise that masked the lower frequency thresholds.

Effect of arterial proteoglycans and glycosaminoglycans on low density lipoprotein oxidation and its uptake by human macrophages and arterial smooth muscle cells.

The reversible interaction of low density lipoprotein (LDL) with arterial chondroitin sulfate proteoglycans (CSPGs) or glycosaminoglycans (GAGs) selects LDL particles with a high affinity for sulfated GAGs and also induces modifications in apolipoprotein B (apo B) and the lipid organization of the lipoprotein. In the present work we studied the effect that the reversible interaction with sulfated polysaccharides has on the susceptibility of LDL to in vitro oxidation. For this purpose soluble, nonaggregated CSPG- or GAG-treated LDL was subjected to oxidation in the presence of 5 microM CuSO4 for as long as 48 hours. The rate of formation of thiobarbituric acid-reactive substances, the decrease in isoelectric point, the increase in relative electrophoretic mobility of LDL, the higher degradation rate by human macrophages, and the lower degradation rate by human arterial smooth muscle cells showed that LDLs exposed to CSPGs and GAGs were significantly more susceptible to oxidation than native LDL. Results from competition experiments indicate that C6S-treated LDL after 4 hours of oxidation is taken up via the acetylated LDL receptor in human macrophages. Coincubation of lipoproteins with human macrophages or human arterial smooth muscle cells for 24 hours also indicated that C6S-treated LDL was more susceptible to cell-induced modifications than native LDL. The occurrence in vivo of similar processes may contribute to focal retention, increased rate oxidation of LDL in the arterial intima, and foam cell formation during atherogenesis.