Persistence of Dichelobacter nodosus, the causal agent of ovine footrot

Ovine footrot (FR) is an economically important disease that causes lameness and affects sheep flocks worldwide. It is characterized by interdigital skin inflammation (interdigital dermatitis [ID]) with, or without, separation of the hoof horn from the underlying tissue (severe footrot [SFR]). The primary causative agent is the gram-negative anaerobic bacterium Dichelobacter nodosus, which is present in diseased feet and thought to be transmitted via contaminated surfaces. Periods of apparent zero prevalence of FR in a flock can be followed by disease occurrence when the climate becomes favourable for pathogen transmission. This suggests that there are sites where D. nodosus persists in the absence of disease. These sites might include healthy feet, the gingival cavity and faeces of sheep and also the environment. The aim of this thesis was to investigate persistence of D. nodosus, by investigating possible sites of survival of D. nodosus over time. Prospective longitudinal studies were used to investigate persistence. Samples were collected from sheep and from the pasture in three studies (Studies 1 and 2: England, study 3: Spain). Quantitative PCR was used to detect and quantify D. nodosus and to investigate associations between D. nodosus presence in feet, in the gingival cavity and on pasture and a range of predictor variables including climate. A multiple locus variable number tandem repeat analysis (MLVA) suitable for use on mixed DNA and environmental samples was optimized and validated to investigate D. nodosus strains within and between sites. A novel approach to characterize individual strains in a sample was designed. D. nodosus was detected in all sample types in all studies but not on all occasions. The feet of sheep were the only site where D. nodosus was detected in loads exceeding 103 cells per swab. In study 1, D. nodosus was detected in amounts exceeding103 cells in samples collected from the pasture in week 1 only, when detection frequency of D. nodosus on feet was high and the weather was wet. A minimum of 14 strains of D. nodosus were detected on the feet of sheep by MLVA. A decline in detection of D. nodosus in the environment coincided with periods of dry weather, however, dry weather did not coincide with a decline in D. nodosus loads on feet or incidence of disease. D. nodosus was more likely to be detected in the gingival cavity when a sheep had FR. It was detected in 25 % of gingival cavity samples and strain types identified in the gingival cavity were the same as the dominant strain types on the feet of sheep. In study 2, disease prevalence and D. nodosus detection frequencies were lower than in study 1. When sheep from the study group were separated from the main flock in week 1 and moved onto pasture that had been unoccupied for 10 days, D. nodosus was transferred to the study group on healthy feet. One dominant strain of D. nodosus persisted throughout an episode of disease and this strain was present on the healthy feet of sheep until up to 5 weeks before the development of lesions in high bacterial loads. There was a reduction in lesion severity and reduced detection of D. nodosus in soil in a period of dry weather. Only 1 sample from the gingival cavity was positive for D. nodosus. Two faecal sample were positive for D. nodosus, indicating for the first time that faecal shedding is possible. In study 3, there were high loads of D. nodosus on healthy feet of a sheep that was classed as susceptible when there had been no cases of FR for at least 2 month. D. nodosus was still present in the flock during the long non-transmission period in the summer. We conclude that D. nodosus is more likely to persist on the feet of sheep, whereas long-term environmental reservoirs of D. nodosus are unlikely. Future research should focus on the feet of sheep and possibly faeces as possible sites of persistence of D. nodosus in the absence of disease

Flotillins Are Involved in the Polarization of Primitive and Mature Hematopoietic Cells

BACKGROUND: Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present evidence that raft-associated endocytic proteins (flotillins) are pre-assembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions. CONCLUSIONS: Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization

Identifying associations between management practices and antimicrobial resistances of sentinel bacteria recovered from bulk tank milk on dairy farms

There is increasing emphasis on the need to reduce antimicrobial use (AMU) on dairy farms to reduce the emergence of resistant bacteria which could compromise animal health and impact human medicine. In addition to AMU, the role of farm management is an area of growing interest and represents an alternative route for possible interventions. The aim of this study was to evaluate the impact of farm management practices and AMU on resistances of sentinel bacteria in bulk milk. Dairy farms from two, geographically separate locations within the British Isles were recruited as part of two study groups. Farm management data from study group 1 (n = 125) and study group 2 (n = 16) were collected by means of a face-to-face questionnaire with farmers carried out during farm visits. For study group 2, additional data on AMU was collated from veterinary medicine sales records. Sentinel bacterial species (Enterococcus spp. and E. coli), which have been reported to be of value in antimicrobial resistance (AMR) studies, were isolated from bulk tank milk to monitor antimicrobial susceptibilities by means of minimum inhibitory concentrations (MICs). MIC data for both groups was used to generate an overall “score” for each farm. For both groups, this overall farm mean MIC was used as the outcome variable to evaluate the impact of farm management and AMU. This was achieved through use of elastic net modelling, a regularised regression method which also featured a bootstrapping procedure to produce robust models. Inference of models was based on covariate stabilities and bootstrapped P-values to identify farm management and AMU practices that have significant effects on MICs of sentinel bacteria. Practices which were found to be of importance with respect to Enterococcus spp. included management of slurry, external entry of livestock to the dairy herd, use of bedding materials and conditioners, cubicle cleaning routines and antibiotic practices, including use of β-lactams and fluoroquinolones. Practices deemed to be of importance for E. coli MICs included cubicle and bedding management practices, teat preparation routines at milking and the milking procedure itself. We conclude that a variety of routine farm management practices are associated with MICs of sentinel bacteria in bulk milk. Amendment of these practices offers additional possible routes of intervention, alongside alterations to AMU, to mitigate the emergence and dissemination of AMR on dairy farms

Argumentationsanalyse von Kommentaren in einem Forum der BBC zur Unabhängigkeit des Kosovo

Die vorliegende Arbeit geht hervor aus dem Hauptseminar „Argumentationstheorie“, das im Wintersemester 2008/09 am Institut für Linguistik der Universität zu Köln unter der Leitung von PD Dr. Leila Behrens abgehalten wurde. Ziel dieses Seminars war es, ausgehend von traditionellen Begriffen der Rhetorik, Dialektik und Logik, in die Terminologie sowie in zentrale Modelle der zeitgenössischen Argumentationsforschung einzuführen. Die dabei erworbenen Kenntnisse sollen im Folgenden bei der Analyse von Beiträgen eines Diskussionsforums im Internet angewendet werden. Hierbei handelt es sich um ein sogenanntes „newsforum“ der BBC mit dem Titel „Have Your Say“ (BBC 2008), in dem aktuelle Themen und Nachrichten von Internetnutzern weltweit diskutiert werden können. Im untersuchten Fall behandeln wir die Frage, wie mit der Unabhängigkeitserklärung des Kosovo vom 17. Februar 2008 umzugehen sei: „Should the world recognise an independent Kosovo?“ […]. Zu dieser Fragestellung wurden insgesamt 3195 Beiträge im Forum veröffentlicht, von denen hier 780 ausgewertet werden. Diese folgen chronologisch aufeinander und umfassen den Zeitraum zwischen 7:49 Uhr (mittlere Greenwich-Zeit) und 14:26 Uhr des 17. Februar 2008

Mass spectrometry and machine learning for the accurate diagnosis of benzylpenicillin and multidrug resistance of Staphylococcus aureus in bovine mastitis

Staphylococcus aureus is a serious human and animal pathogen threat exhibiting extraordinary capacity for acquiring new antibiotic resistance traits in the pathogen population worldwide.The development of fast, affordable and effective diagnostic solutions capable of discriminating between antibiotic-resistant and susceptible S. aureus strains would be of huge benefit for effective disease detection and treatment. Here we develop a diagnostics solution that uses Matrix-Assisted Laser Desorption/Ionisation–Time of Flight Mass Spectrometry (MALDI-TOF) and machine learning, to identify signature profiles of antibiotic resistance to either multidrug or benzylpenicillin in S. aureus isolates. Using ten different supervised learning techniques, we have analysed a set of 82 S. aureus isolates collected from 67 cows diagnosed with bovine mastitis across 24 farms. For the multidrug phenotyping analysis, LDA, linear SVM, RBF SVM, logistic regression, naïve Bayes, MLP neural network and QDA had Cohen’s kappa values over 85.00%. For the benzylpenicillin phenotyping analysis, RBF SVM, MLP neural network, naïve Bayes, logistic regression, linear SVM, QDA, LDA, and random forests had Cohen’s kappa values over 85.00%. For the benzylpenicillin the diagnostic systems achieved up to (mean result ± standard deviation over 30 runs on the test set): accuracy = 97.54% ± 1.91%, sensitivity = 99.93% ± 0.25%, specificity = 95.04% ± 3.83%, and Cohen’s kappa = 95.04% ± 3.83%. Moreover, the diagnostic platform complemented by a protein-protein network and 3D structural protein information framework allowed the identification of five molecular determinants underlying the susceptible and resistant profiles. Four proteins were able to classify multidrug-resistant and susceptible strains with 96.81% ± 0.43% accuracy. Five proteins, including the previous four, were able to classify benzylpenicillin resistant and susceptible strains with 97.54% ± 1.91% accuracy. Our approach may open up new avenues for the development of a fast, affordable and effective day-to-day diagnostic solution, which would offer new opportunities for targeting resistant bacteria

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addresse

Biological properties of extracellular vesicles and their physiological functions

María Yáñez-Mó#, Pia R.-M. Siljander#, Zoraida Andreu, Apolonija Bedina Zavec, Francesc E. Borràs, Edit I. Buzas, Krisztina Buzas, Enriqueta Casal, Francesco Cappello, Joana Carvalho, Eva Colás, Anabela Cordeiro-da Silva, Stefano Fais, Juan M. Falcon-Perez, Irene M. Ghobrial, Bernd Giebel, Mario Gimona, Michael Graner, Ihsan Gursel, Mayda Gursel, Niels H. H. Heegaard, An Hendrix30, Peter Kierulf, Katsutoshi Kokubun, Maja Kosanovic, Veronika Kralj-Iglic, Eva-Maria Krämer-Albers, Saara Laitinen, Cecilia Lässer, Thomas Lener, Erzsébet Ligeti, Aija Linē, Georg Lipps, Alicia Llorente, Jan Lötvall, Mateja Manček-Keber, Antonio Marcilla, Maria Mittelbrunn, Irina Nazarenko, Esther N.M. Nolte-‘t Hoen, Tuula A. Nyman, Lorraine O'Driscoll, Mireia Olivan, Carla Oliveira, Éva Pállinger, Hernando A. del Portillo, Jaume Reventós, Marina Rigau, Eva Rohde, Marei Sammar, Francisco Sánchez-Madrid, N. Santarém1, Katharina Schallmoser, Marie Stampe Ostenfeld, Willem Stoorvogel, Roman Stukelj, Susanne G. Van der Grein, M. Helena Vasconcelos, Marca H. M. Wauben and Olivier De WeverIn the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells.While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.Peer reviewe

Mesenchymal stem cells augment the anti-bacterial activity of neutrophil granulocytes.

BackgroundMesenchymal stem cells (MSCs) participate in the regulation of inflammation and innate immunity, for example by responding to pathogen-derived signals and by regulating the function of innate immune cells. MSCs from the bone-marrow and peripheral tissues share common basic cell-biological functions. However, it is unknown whether these MSCs exhibit different responses to microbial challenge and whether this response subsequently modulates the regulation of inflammatory cells by MSCs.Methodology/principal findingsWe isolated MSCs from human bone-marrow (bmMSCs) and human salivary gland (pgMSCs). Expression levels of TLR4 and LPS-responsive molecules were determined by flow cytometry and quantitative PCR. Cytokine release was determined by ELISA. The effect of supernatants from unstimulated and LPS-stimulated MSCs on recruitment, cytokine secretion, bacterial clearance and oxidative burst of polymorphonuclear neutrophil granulocytes (PMN) was tested in vitro. Despite minor quantitative differences, bmMSCs and pgMSCs showed a similar cell biological response to bacterial endotoxin. Both types of MSCs augmented anti-microbial functions of PMNs. LPS stimulation, particularly of bmMSCs, further augmented MSC-mediated activation of PMN [corrected].Conclusions/significanceThis study suggests that MSCs may contribute to the resolution of infection and inflammation by promoting the anti-microbial activity of PMNs. This property is exerted by MSCs derived from both the bone-marrow and peripheral glandular tissue