A cost-benefit assessment of Salmonella-control strategies in pigs reared in the United Kingdom.

Pork and pork products are a major source of human salmonellosis in the United Kingdom (UK). Despite a number of surveillance programmes, the prevalence of Salmonella in the UK slaughter pig population remains over 20%. Here, we present the results of a Cost-Benefit Analysis comparing five on-farm control strategies (where the cost is the cost of implementation and the benefits are the financial savings for both the human health and pig industries). The interventions considered were: wet feed, organic acids in feed, vaccination, enhanced cleaning and disinfection and movement of outdoor breeding units. The data originate from published papers and recent UK studies. The effectiveness was assessed by adapting a previous risk assessment, originally developed for the European Food Safety Authority. Using this method, none of the intervention strategies produced a net cost-benefit. Our results suggest that the cost of implementation outweighed the savings for all interventions, even if the effectiveness could be improved. Therefore, to achieve a net cost-benefit it is essential to reduce the cost of interventions. Analyses concluded that large cost reductions (up to 96%) would be required. Use of organic acids required the smallest reduction in cost (22.7%) to achieve a net cost benefit. Uncertainty analysis suggested that a small net gain might be possible, for some of the intervention measures. But this would imply that the model greatly underestimated some key parameters, which was considered unlikely. Areas of key uncertainty were identified as the under-reporting factor (i.e. the proportion of community cases of Salmonella) and the source attribution factor (i.e. the proportion of human Salmonella cases attributable to pork products).

Towards an integrated food safety surveillance system: a simulation study to explore the potential of combining genomic and epidemiological metadata.

Foodborne infection is a result of exposure to complex, dynamic food systems. The efficiency of foodborne infection is driven by ongoing shifts in genetic machinery. Next-generation sequencing technologies can provide high-fidelity data about the genetics of a pathogen. However, food safety surveillance systems do not currently provide similar high-fidelity epidemiological metadata to associate with genetic data. As a consequence, it is rarely possible to transform genetic data into actionable knowledge that can be used to genuinely inform risk assessment or prevent outbreaks. Big data approaches are touted as a revolution in decision support, and pose a potentially attractive method for closing the gap between the fidelity of genetic and epidemiological metadata for food safety surveillance. We therefore developed a simple food chain model to investigate the potential benefits of combining 'big' data sources, including both genetic and high-fidelity epidemiological metadata. Our results suggest that, as for any surveillance system, the collected data must be relevant and characterize the important dynamics of a system if we are to properly understand risk: this suggests the need to carefully consider data curation, rather than the more ambitious claims of big data proponents that unstructured and unrelated data sources can be combined to generate consistent insight. Of interest is that the biggest influencers of foodborne infection risk were contamination load and processing temperature, not genotype. This suggests that understanding food chain dynamics would probably more effectively generate insight into foodborne risk than prescribing the hazard in ever more detail in terms of genotype.

A Transport and Lairage Model for Salmonella Transmission Between Pigs Applicable to EU Member States.

A model for the transmission of Salmonella between finisher pigs during transport to the abattoir and subsequent lairage has been developed, including novel factors such as environmental contamination and the effect of stress, and is designed to be adaptable for any EU Member State (MS). The model forms part of a generic farm-to-consumption model for Salmonella in pigs, designed to model potentially important risk factors and assess the effectiveness of interventions. In this article, we discuss the parameterization of the model for two case study MSs. For both MSs, the model predicted an increase in the average MS-level prevalence of Salmonella-positive pigs during both transport and lairage, accounting for a large amount of the variation between reported on-farm prevalence and reported lymph-node prevalence at the slaughterhouse. Sensitivity analysis suggested that stress is the most important factor during transport, while a number of factors, including environmental contamination and the dose-response parameters, are important during lairage. There was wide variation in the model-predicted change in prevalence in individual batches; while the majority of batches (80-90%) had no increase, in some batches the increase in prevalence was over 70% and in some cases infection was introduced into previously uninfected batches of pigs. Thus, the model suggests that while the transport and lairage stages of the farm-to-consumption exposure pathway are unlikely to be responsible for a large increase in average prevalence at the MS level, they can have a large effect on prevalence at an individual-batch level.

Modelling the species jump: towards assessing the risk of human infection from novel avian influenzas.

The scientific understanding of the driving factors behind zoonotic and pandemic influenzas is hampered by complex interactions between viruses, animal hosts and humans. This complexity makes identifying influenza viruses of high zoonotic or pandemic risk, before they emerge from animal populations, extremely difficult and uncertain. As a first step towards assessing zoonotic risk of influenza, we demonstrate a risk assessment framework to assess the relative likelihood of influenza A viruses, circulating in animal populations, making the species jump into humans. The intention is that such a risk assessment framework could assist decision-makers to compare multiple influenza viruses for zoonotic potential and hence to develop appropriate strain-specific control measures. It also provides a first step towards showing proof of principle for an eventual pandemic risk model. We show that the spatial and temporal epidemiology is as important in assessing the risk of an influenza A species jump as understanding the innate molecular capability of the virus. We also demonstrate data deficiencies that need to be addressed in order to consistently combine both epidemiological and molecular virology data into a risk assessment framework.

Extracellular fluid conductivity analysis by dielectric spectroscopy for in vitro determination of cortical tissue vitality.

Brain tissue is extremely metabolically active in part due to its need to constantly maintain a precise extracellular ionic environment. Under pathological conditions, unhealthy cortical tissue loses its ability to maintain this precise environment and there is a net efflux of charged particles from the cells. Typically, this ionic efflux is measured using ion-selective microelectrodes, which measure a single ionic species at a time. In this paper, we have used a bio-sensing method, dielectric spectroscopy (DS), which allows for the simultaneous measurement of the net efflux of all charged particles from cells by measuring extracellular conductivity. We exposed cortical brain slices from the mouse to different solutions that mimic various pathological states such as hypokalemia, hyperkalemia and ischemia (via oxygen-glucose deprivation). We have found that the changes in conductivity of the extracellular solutions were proportional to the severity of the pathological insult experienced by the brain tissue. Thus, DS allows for the measurement of changes in extracellular conductivity with enough sensitivity to monitor the health of brain tissue in vitro.

Design and implementation of a novel superfusion system for ex vivo characterization of neural tissue by dielectric spectroscopy (DS).

Dielectric spectroscopy is a widely utilized electrophysiological characterization method. The obtained dielectric spectra and derived properties have the potential of providing significant information regarding changes in the physiological state of a biological system. However, since many of the dielectric properties are obtained in vitro from excised tissue far removed from physiological conditions, the value of the information obtained may be diminished. In this paper, we introduce a superfusion system that is designed to produce ex vivo dielectric spectroscopy measurements by providing the living tissue with a continuous and ample supply of nutrients and oxygen while removing metabolites and other waste. This superfusion system provides the convenience of in vitro measurement while concurrently producing results that can be more closely correlated with actual physiological changes in the biological system.

Dynamics of Salmonella transmission on a British pig grower-finisher farm: a stochastic model.

Previous modelling studies have estimated that between 1% and 10% of human salmonella infections are attributable to pig meat consumption. In response to this food safety threat the British pig industry have initiated a salmonella monitoring programme. It is anticipated that this programme will contribute to achieving a UK Food Standards Agency target for reducing salmonella levels in pigs at slaughter by 50% within 5 years. In order to better inform the monitoring programme, we have developed a stochastic transmission model for salmonella in a specialist grower-finisher pig herd, where data from a Danish longitudinal study have been used to estimate some of the key model parameters. The model estimates that about 17% of slaughter-age pigs will be infected with salmonella, and that of these infected pigs about 4% will be excreting the organism. In addition, the model shows that the most effective control strategies will be those that reduce between-pen transmission.

Rat supraspinatus tendon expresses cartilage markers with overuse.

The goals of this study were to investigate the response of the rat supraspinatus tendon to overuse at the molecular level using transcriptional profiling, and to identify potential markers of tendinopathy. Adult rats were subjected to an overuse protocol that consists of downhill running (10% grade) at 17 m/min for 1 h/day, 5 days/week, for a total of either 1, 2, or 4 weeks. Another group of rats served as nonrunning time 0 controls. Transcriptional profiling was performed on the supraspinatus and patellar tendons using an Affymetrix rat genome array. A gene was considered to be differentially expressed if the p value from an ANOVA test was less than 0.01 and the difference between runners and controls was at least twofold at any time point. The supraspinatus tendon had increased expression of well-known cartilage genes such as col2a1, aggrecan, and sox9. These genes were not regulated in the patellar tendon, an internal comparator. Few genes associated with inflammation, or angiogenesis, were differentially expressed, and no significant change in the regulation of matrix metalloproteinases was detected. The results of this study suggest that by expressing more cartilage genes, the tendon is converting toward a fibrocartilage phenotype as a result of the repetitive loading and repeated compression of the tendon as it passes through the acromial arch.

Gene expression analysis in a murine model of allergic asthma reveals overlapping disease and therapy dependent pathways in the lung.

Accumulating evidence in animal models and human asthma support a central role for IL-13 signaling in disease pathogenesis. In order to identify asthma and therapy associated genes, global transcriptional changes were monitored in mouse lung following antigen challenge (ovalbumin (OVA)), either alone or in the presence of a soluble IL-13 antagonist. Changes in whole lung gene expression after instillation of mIL-13 were also measured both in wild type and STAT6 deficient mice. A striking overlap in the gene expression profiles induced by either OVA challenge or mIL-13 was observed, further strengthening the relationship of IL-13 signaling to asthma. Consistent with results from functional studies, a subset of the OVA-induced gene expression was significantly inhibited by a soluble IL-13 antagonist while IL-13-modulated gene expression was completely attenuated in the absence of STAT6-mediated signaling. Results from these experiments greatly expand our understanding of asthma and provide novel molecular targets for therapy and potential biomarkers of IL-13 antagonism.

A model of a segmental oscillator in the leech heartbeat neuronal network.

We modeled a segmental oscillator of the timing network that paces the heartbeat of the leech. This model represents a network of six heart interneurons that comprise the basic rhythm-generating network within a single ganglion. This model builds on a previous two cell model (Nadim et al., 1995) by incorporating modifications of intrinsic and synaptic currents based on the results of a realistic waveform voltage-clamp study (Olsen and Calabrese, 1996). Due to these modifications, the new model behaves more similarly to the biological system than the previous model. For example, the slow-wave oscillation of membrane potential that underlies bursting is similar in form and amplitude to that of the biological system. Furthermore, the new model with its expanded architecture demonstrates how coordinating interneurons contribute to the oscillations within a single ganglion, in addition to their role of intersegmental coordination.

Quantitative analysis of mRNA amplification by in vitro transcription.

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

Evaluation of normalization procedures for oligonucleotide array data based on spiked cRNA controls.

BACKGROUND: Affymetrix oligonucleotide arrays simultaneously measure the abundances of thousands of mRNAs in biological samples. Comparability of array results is necessary for the creation of large-scale gene expression databases. The standard strategy for normalizing oligonucleotide array readouts has practical drawbacks. We describe alternative normalization procedures for oligonucleotide arrays based on a common pool of known biotin-labeled cRNAs spiked into each hybridization. RESULTS: We first explore the conditions for validity of the 'constant mean assumption', the key assumption underlying current normalization methods. We introduce 'frequency normalization', a 'spike-in'-based normalization method which estimates array sensitivity, reduces background noise and allows comparison between array designs. This approach does not rely on the constant mean assumption and so can be effective in conditions where standard procedures fail. We also define 'scaled frequency', a hybrid normalization method relying on both spiked transcripts and the constant mean assumption while maintaining all other advantages of frequency normalization. We compare these two procedures to a standard global normalization method using experimental data. We also use simulated data to estimate accuracy and investigate the effects of noise. We find that scaled frequency is as reproducible and accurate as global normalization while offering several practical advantages. CONCLUSIONS: Scaled frequency quantitation is a convenient, reproducible technique that performs as well as global normalization on serial experiments with the same array design, while offering several additional features. Specifically, the scaled-frequency method enables the comparison of expression measurements across different array designs, yields estimates of absolute message abundance in cRNA and determines the sensitivity of individual arrays.

Genomic analysis of gene expression in C. elegans.

Until now, genome-wide transcriptional profiling has been limited to single-cell organisms. The nematode Caenorhabditis elegans is a well-characterized metazoan in which the expression of all genes can be monitored by oligonucleotide arrays. We used such arrays to quantitate the expression of C. elegans genes throughout the development of this organism. The results provide an estimate of the number of expressed genes in the nematode, reveal relations between gene function and gene expression that can guide analysis of uncharacterized worm genes, and demonstrate a shift in expression from evolutionarily conserved genes to worm-specific genes over the course of development.

Multiple differences in gene expression in regulatory Valpha 24Jalpha Q T cells from identical twins discordant for type I diabetes.

Quantitative and qualitative defects in CD1d-restricted T cells have been demonstrated in human and murine autoimmune diseases. To investigate the transcriptional consequences of T cell receptor activation in human Valpha24JalphaQ T cell clones, DNA microarrays were used to quantitate changes in mRNA levels after anti-CD3 stimulation of clones derived from identical twins discordant for type 1 diabetes and IL-4 secretion. Activation resulted in significant modulation of 226 transcripts in the IL-4 secreting clone and 86 in the IL-4-null clone. Only 28 of these genes were in common. The differences observed suggest both ineffective differentiation of diabetic Valpha24JalphaQ T cells and a role for invariant T cells in the recruitment and activation of cells from the myeloid lineage.

Evidence for two forms of substructure in the cell survival curve--mechanisms and clinical consequences.

Recent laboratory studies have clearly demonstrated the presence of two types of fine structure in the radiation survival response of cultured mammalian cells: a) one type of substructure, observed at doses of a few Gy, is the result of the differential killing of subpopulations of cells of different, cell-cycle-related radiosensitivity; this substructure is strongly dependent on the cell-cycle distribution and is absent in tightly synchronized cell populations; b) the other type of substructure, found at lower doses (< 1 Gy), is expressed as a very sensitive (hypersensitive) response at very low doses followed by increased resistance as the dose increases until, by approximately 1 Gy, the response usually follows a standard linear-quadratic (LQ) function; it thus has the characteristics of a radiation-induced radioresistance and is assumed to reflect an inducible repair process. Although the linear-quadratic (LQ) model is widely used to describe the dose-effect response both in the laboratory and in the clinic, over the past 20 years there have been several reports of an anomalous departure from the simple LQ formalism, particularly at low doses. A review of these reports suggests that the observed anomalies are not so much a failure of the LQ formalism as a manifestation of the effects of the response substructure: mixed populations, a) and hypersensitivity, b) described above.

Lack of correlation between G1 arrest and radiation age-response in three synchronized human tumour cell lines.

PURPOSE: To determine radiosensitivity as a function of cell age (the age-response) in three human tumour cell lines, and investigate the dependence of the age-response on G1 arrest and on cell-age heterogeneity in synchronized cell populations. MATERIALS AND METHODS: Variation in radiosensitivity throughout the cell cycle and G1 arrest was measured in mitotically selected populations of synchronized human tumour cells. In order to examine the effects of desynchronization and cell age heterogeneity on the measured age-response, a mathematical model was developed based on an existing kinetic model of the cell cycle. The model was used to describe the age-response for mitotically selected populations of cells, which was then compared with experimentally measured age responses. RESULTS: Three different human tumour cell lines had qualitatively similar age-responses, with periods of radiosensitivity in mitosis and in late G1 phase/early S phase, and periods of radioresistance in early/mid G1 phase and late S/G2 phase. Radiosensitivity appeared to increase in G1 phase before the onset of DNA synthesis. One of the cell lines displayed a prolonged G1 arrest after irradiation in G1 phase. Model results demonstrated that the measured age-responses were consistent with a simple model in which the cell cycle was divided into four regions. Radiosensitivity was assumed to be constant within each region, and changed abruptly at the borders between regions. CONCLUSIONS: Human tumour cell lines can exhibit qualitatively similar age-responses despite having markedly different G1 checkpoint responses. This suggests that modulation of the G1 arrest response may not prove to be a useful clinical strategy because it may not lead to significant cell age specific changes in radiosensitivity. The mathematical model of the radiation response of mitotically selected synchronized cells was a useful way to quantitatively describe cell age heterogeneity in these populations, and demonstrated the important impact of this heterogeneity on measured age-responses.

Cell-age heterogeneity and deviations from the LQ model in the radiation survival responses of human tumour cells.

PURPOSE: To determine whether some of the deviations from the simple linear-quadratic (LQ) theory in the radiation dose survival responses of asynchronous cultures of human tumour cell lines are caused by the presence of cell-age specific subpopulations which all individually follow LQ theory, but have different radiosensitivities. MATERIALS AND METHODS: Human tumour cells were synchronized by mitotic selection and their survival dose responses were measured at doses from 0.05 Gy to 12 Gy, using a high-precision survival assay. These responses were compared with a kinetic model of radiation survival in synchronized cells, which assumed that age-specific populations individually obeyed the LQ theory. The cell lines used included HT-29 and A549, which have typical dose responses, and U1, which is somewhat atypical. RESULTS: In two of the three cell lines, A549 and HT-29, observed deviations from the LQ model were consistent with those expected from cell-age heterogeneity. In the third cell line, U1, survival responses could not be described by the LQ theory, even when cell-age heterogeneity was considered. CONCLUSIONS: The LQ model provided an adequate description of cell survival for two of three tumour cell lines in this study when cell-age related heterogeneity in survival responses was accounted for. However, some alternative survival models (such as the repair saturation model) provided better characterizations of the survival response of the third cell line and, in fact, gave good descriptions of survival for all three cell lines.

Effect of a chimeric antibody to tumor necrosis factor-alpha on cytokine and physiologic responses in patients with severe sepsis--a randomized, clinical trial.

OBJECTIVES: Tumor necrosis factor (TNF)-alpha appears central to the pathogenesis of severe sepsis, but aspects of the cytokine cascade and the link to physiologic responses are poorly defined. We hypothesized that a monoclonal antibody to TNF-alpha given early in the course of severe sepsis would modify the pattern of systemic cytokine release and, as a consequence, resuscitation fluid requirements, net proteolysis, and hypermetabolism would be reduced. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: Critical Care Unit and University Department of Surgery in a single tertiary care center. PATIENTS: Fifty-six patients (from 92 eligible patients) with severe sepsis. Twenty-eight patients were randomized to treatment, and were comparable with the placebo group for age, gender, race, Acute Physiology and Chronic Health Evaluation II score, and site and type of infection. INTERVENTIONS: A 300-mg single dose of cA2 (a chimeric neutralizing antibody to TNF-alpha) was given intravenously within 12 hrs of the onset of severe sepsis. Standard surgical and intensive care therapy was otherwise delivered. MEASUREMENTS AND MAIN RESULTS: Plasma concentrations of TNF-alpha, interleukin (IL)-1beta IL-6, IL-8, IL-10, soluble 75-kilodalton TNF-alpha receptor (sTNFR-75), and IL-1beta receptor antagonist (IL-1ra) were measured by sandwich enzyme-linked immunosorbent assay before cA2 infusion, 8 hrs later, and then daily for a minimum of 4 days. Sequential changes in total body protein, body water spaces, and resting energy expenditure over 21 days were measured, as soon as patients achieved hemodynamic stability, by in vivo neutron activation analysis, tritium and bromide dilution, and indirect calorimetry, respectively. Twenty-one patients died, ten having received cA2. Suppression of measurable TNF-alpha was observed at 8 hrs with subsequent rebound by 24 hrs after cA2 treatment. The concentrations of other cytokines were high, were not reduced by intervention, and decreased logarithmically over 5 days. Both groups reached hemodynamic stability at similar times (57.5 +/- 11.8 hrs in controls vs. 58.6 +/- 9.2 hrs in the cA2 group) and following similar volumes of infused fluids (29.1 +/- 3.4 L vs. 28.9 +/- 4.4 L). No differences in net proteolysis, resolution of body water expansion, or alteration in resting energy expenditure were demonstrated. CONCLUSION: A single dose of cA2 did not alter the overall pattern of cytokine activation or the profound derangements in physiologic function that accompany severe sepsis.

Regulation of synaptic depression rates in the cricket cercal sensory system.

To assess the roles of pre- and postsynaptic mechanisms in the regulation of depression, short-term synaptic depression was characterized at the synapses between sensory neurons and two interneurons in the cricket cercal sensory system. Changes in excitatory postsynaptic potential (EPSP) amplitude with repetitive stimulation at 5 and 20 Hz were quantified and fitted to the depletion model of transmitter release. The depression rates of different sensory neuron synapses on a single interneuron varied with the age of the sensory neurons such that old sensory neuron synapses depressed faster than young synapses. Although all synapses showed depression, short-term facilitation was selectively expressed only at sensory neuron synapses on one interneuron, the medial giant interneuron (MGI). These synapses showed concurrent facilitation and depression with high-frequency stimulation (100 Hz), whereas the synapses on another interneuron, 10-3, showed only depression at all stimulus frequencies. A previous study showed that the ability of a synapse to facilitate is correlated with the identity of the postsynaptic neuron. The present results indicate that depression and facilitation are regulated independently. Depression is regulated presynaptically in a manner related to sensory neuron age; whereas, facilitation is regulated by the postsynaptic target.

Massive nitrogen loss in critical surgical illness: effect on cardiac mass and function.

OBJECTIVE: The authors measured cardiac mass and function to determine whether these changed in patients who were critically ill who were losing large amounts of nitrogen from the body. SUMMARY BACKGROUND DATA: The large losses of body nitrogen that occur in patients with protein-energy malnutrition are associated with a loss of cardiac mass and function. It is not known if this also occurs in patients who were critically ill who are losing massive amounts of nitrogen. METHODS: Once hemodynamically stable, 13 patients who were critically ill underwent sequential measurements of left ventricular mass (LVM) and function, total body nitrogen (TBN), total body potassium, body weight, fat-free mass, and limb muscle mass. RESULTS: Over a 21-day study period, there was no change in LVM or function despite falls of 14% and 21% in TBN and total body potassium, respectively, a 21% fall in limb muscle mass, and a deterioration in skeletal muscle function by approximately 40%. CONCLUSIONS: In patients who were critically ill, cardiac mass does not decrease and function does not deteriorate after hemodynamic stability has been achieved despite massive losses of protein from the body.