Explorative Field Study on the Use of Oral Fluids for the Surveillance of Actinobacillus pleuropneumoniae Infections in Fattening Farms by an Apx-Real-Time PCR

Simple Summary Oral fluid sampling (OFS) is an animal friendly and easy way for surveillance purposes in domestic swine populations, especially concerning respiratory diseases. In case of Actinobacillus (A.) pleuropneumoniae surveillance, measures are usually combined with burdensome sampling for animals and humans. In the present study, we evaluated the suitability of oral fluids (OFs) for surveillance purposes of A. pleuropneumoniae infections in fattening pigs using an Apx-toxin real-time PCR. We were able to demonstrate that the examination of OFs by an Apx-toxin real-time PCR is suitable for A. pleuropneumoniae surveillance in the field in an animal friendly and easy way. These results might contribute to an increased compliance of laboratory diagnostic measures on pig farms and thereby to increased animal welfare due to less burdensome sampling and improved animal health. Oral fluids (OFs) represent a cost effective and reliable tool for surveillance purposes, mostly regarding viruses. In the present study, we evaluated the suitability of OFs for surveillance purposes concerning Actinobacillus (A.) pleuropneumoniae infections in fattening pigs under field conditions. OFs were examined with an Apx-toxin real-time PCR that detects the genes encoding for Apx I-, Apx III-, and Apx IV-toxin. For this purpose, we conducted a pen-wise collection of OFs over one fattening period from fattening pigs of two farms (farm A and B) with a known history of A. pleuropneumoniae infection. Lung lesions were determined at slaughter to estimate the extend of pulmonary lesions and pleural affection. Apx III- and Apx IV-toxin DNA were present in the OFs of both farms whereas Apx I-toxin DNA was present on farm A only. We were able to detect Apx I-, Apx III-, and Apx IV-toxin DNA in different patterns directly after introduction of the new pigs in the farms and over the entire study period. In summary, or results indicate the suitability of OFS for the early detection and surveillance of A. pleuropneumoniae in fattening farms

A modified agar pad method for mycobacterial live-cell imaging

<p>Abstract</p> <p>Background</p> <p>Two general approaches to prokaryotic live-cell imaging have been employed to date, growing bacteria on thin agar pads or growing bacteria in micro-channels. The methods using agar pads 'sandwich' the cells between the agar pad on the bottom and a glass cover slip on top, before sealing the cover slip. The advantages of this technique are that it is simple and relatively inexpensive to set up. However, once the cover slip is sealed, the environmental conditions cannot be manipulated. Furthermore, desiccation of the agar pad, and the growth of cells in a sealed environment where the oxygen concentration will be in gradual decline, may not permit longer term studies such as those required for the slower growing mycobacteria.</p> <p>Findings</p> <p>We report here a modified agar pad method where the cells are sandwiched between a cover slip on the bottom and an agar pad on top of the cover slip (rather than the reverse) and the cells viewed from below using an inverted microscope. This critical modification overcomes some of the current limitations with agar pad methods and was used to produce time-lapse images and movies of cell growth for <it>Mycobacterium smegmatis </it>and <it>Mycobacterium bovis </it>BCG.</p> <p>Conclusions</p> <p>This method offers improvement on the current agar pad methods in that long term live cell imaging studies can be performed and modification of the media during the experiment is permitted.</p

Common breast cancer susceptibility alleles are associated with tumor subtypes in BRCA1 and BRCA2 mutation carriers : results from the Consortium of Investigators of Modifiers of BRCA1/2.

Abstract Introduction Previous studies have demonstrated that common breast cancer susceptibility alleles are differentially associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers. It is currently unknown how these alleles are associated with different breast cancer subtypes in BRCA1 and BRCA2 mutation carriers defined by estrogen (ER) or progesterone receptor (PR) status of the tumour. Methods We used genotype data on up to 11,421 BRCA1 and 7,080 BRCA2 carriers, of whom 4,310 had been affected with breast cancer and had information on either ER or PR status of the tumour, to assess the associations of 12 loci with breast cancer tumour characteristics. Associations were evaluated using a retrospective cohort approach. Results The results suggested stronger associations with ER-positive breast cancer than ER-negative for 11 loci in both BRCA1 and BRCA2 carriers. Among BRCA1 carriers, single nucleotide polymorphism (SNP) rs2981582 (FGFR2) exhibited the biggest difference based on ER status (per-allele hazard ratio (HR) for ER-positive = 1.35, 95% CI: 1.17 to 1.56 vs HR = 0.91, 95% CI: 0.85 to 0.98 for ER-negative, P-heterogeneity = 6.5 &#215; 10-6). In contrast, SNP rs2046210 at 6q25.1 near ESR1 was primarily associated with ER-negative breast cancer risk for both BRCA1 and BRCA2 carriers. In BRCA2 carriers, SNPs in FGFR2, TOX3, LSP1, SLC4A7/NEK10, 5p12, 2q35, and 1p11.2 were significantly associated with ER-positive but not ER-negative disease. Similar results were observed when differentiating breast cancer cases by PR status. Conclusions The associations of the 12 SNPs with risk for BRCA1 and BRCA2 carriers differ by ER-positive or ER-negative breast cancer status. The apparent differences in SNP associations between BRCA1 and BRCA2 carriers, and non-carriers, may be explicable by differences in the prevalence of tumour subtypes. As more risk modifying variants are identified, incorporating these associations into breast cancer subtype-specific risk models may improve clinical management for mutation carriers

Temperature measurement with the infrared camera in adult nude mice. Refinement

Etablierte Methoden zur Erfassung der Körpertemperatur sind invasiv, wodurch diese ein Maß an Belastung und Stress für Mäuse verursachen. Vor diesem Hintergrund wurde die nicht invasive Thermografie, als angewendetes Refinement mit etablierten, invasiveren Temperaturmessverfahren an Nacktmäusen verglichen. In dieser Studie zeigten die qualitative Thermografie von einzelnen Nacktmäusen und von Mäusen in der Gruppe, dass die Methode geeignet ist, die abgestrahlte thermische Energie (Wärmestrahlung) zu messen und physiologische Zustände zu detektieren. Eine akkurate Identifikation aller Mäuse per Wärmebildkamera ist bei größeren Käfiggruppen und mehreren Thermogrammen trotz zusätzlicher digitaler Bildgebung jedoch nicht praktikabel. Die qualitativen Thermogramme der Mäuse in der Gruppe sind geeignet einzelne auffällige Mäuse einer Käfiggruppe wiederholt im Thermogramm zu detektieren. Die abgestrahlte thermische Energie von Nacktmäusen in der Gruppe kann im qualitativen Thermogramm mittels Wärmebildkamera FLIR E40 visualisiert werden. Die quantitative Thermografie stellt eine praktische Anwendung dar, um berührungslos und nicht invasiv, die Temperaturen von Nacktmäusen im physiologischen Bereich reproduzierbar zu messen. Die vorliegende Studie ergab, dass ein Temperaturwert aus einer minimalen Anzahl von 10 Thermogrammen bei der quantitativen Methode genutzt werden sollte. Bezugnehmend auf den Erkenntnissen der quantitativen Thermografie ist davon auszugehen, dass thermografisch gemessene Hauttemperaturen nicht überschätzen werden. Daraus resultiert, dass der höchste, maximal gemessene Temperaturwert (irTmax) aller Thermogramme derjenige Wert ist, der den realen irT-Wert am besten widerspiegelt. Die Studienergebnisse zeigen, dass die höchste irTmax aus allen Thermogrammen bei einer quantitativen Messung als Temperaturwert genutzt werden sollte, statt eines Mittelwertes der Temperaturen oder eines Mittelwertes der maximalen irT (irTmax). Sowohl die thermografischen Messungen der 4 weiblichen BALB/cAnN - Foxn1nu/nu/Rj Mäuse unterschiedlichen Ernährungszustands, als auch die Messung der 50 männlichen Rj:NMRI - Foxn1nu/nu Mäuse und die Mehrfachmessungen der 20 BALB/cAnN - Foxn1nu/nu/Rj Mäuseböcke über einen längeren Zeitraum zeigen die Praktikabilität der Wärmebildkamera. Die für alle quantitativen Thermogramme verwendete Wärmebildkamera des Modells FLIR E40 hat eine höhere Messfleckgröße und eine geringere Kameraauflösung und -qualität, verglichen mit den momentanen technischen Möglichkeiten. Für den Vergleich der verschiedenen Temperaturmessmethoden wurde die Wärmebildkamera des Modells FLIR T660 genutzt. Eine geringere Größe des Messflecks ermöglichte es, mit der WBK FLIR T660 hochwertigere irT - Ergebnisse zu erhalten als bei der Messung mit dem Modell FLIR E40. Eine automatische Fokussierung ist auf Grund des notwendigen Kontrastes in Form eines minimalen Temperaturunterschiedes in einigen Anwendungsgebieten allerding umstritten. In dieser Studie zeigt sich, dass die Autofokusfunktion des Modells FLIR T660 die Nutzung eines Stativs bedingt, um Mikrovibrationen der Hand des Anwenders zu vermeiden und hochwertige Temperaturergebnisse zu erhalten. Nur die Verwendung eines Stativs ermöglicht die Fokussierung einer Nacktmaus in einer konstanten Ebene zur thermografischen Messung. Wobei einzig die hohe Aufnahmefrequenz des Modells FLIR T660 radiometrische autofokussierte Videoaufnahmen selbst bei bewegten Mäusen ermöglicht. In dieser Studie wurde die Anwendung der Wärmebildkamera mit invasiveren Methoden, wie einer rektalen Sonde, einem subkutan implantieren Transponder und einen intraperitoneal implantierten Datenloggen gegenübergestellt. Die Wärmebildkamera ist ein stressfreies Messwerkzeug, sodass kein chirurgischer Eingriff und kein Einbringen von Fremdmaterial in die Maus notwendig sind. Rektal mit der Sonde model 5885, Precision Digital Thermometer with PRT [platinum resistance thermometer] (H Tinsley, New Addington, United Kingdom) erhobene Temperaturwerte sollten vorsichtig interpretiert werden, da die Durchführung zu Komplikationen führen kann und die entstehenden Einzeltiereffekte bei der Fehlerbetrachtung berücksichtigt werden müssen. Die Transponder-Messungen mit den IPTT-100 (Bio Medic Data Systems, Seaford, DE) haben zusätzlich den Vorteil der Möglichkeit einer Identifizierung der Maus. Wobei diese Studie zeigt, dass im Experiment die Nutzung von temperaturkalibrierten Transpondern zu empfehlen ist. Die Datenlogger, DST Nano–T Temperature Recorder (Starr Oddi, Gardabaer, Iceland) haben zwar den Vorteil der kontinuierlichen Temperaturmessung, aber in dieser tierexperimentellen Studie, waren die von der Software berechneten, registrierten intraperitoneal gemessenen Temperaturen für die Fragestellung nicht verwendbar. Die Ergebnisse zeigen, dass die Wärmebildkamera ebenso genau ist wie die rektale und die subkutane Temperaturmessmethode. Die Studie zeigt, dass die infrarot gemessene Temperatur geeignet ist, die Temperatur von Nacktmäusen widerzuspiegeln und ebenso valide ist wie die rektale und subkutane Temperaturmessung. Zusammengefasst stellt die höchst effiziente, nicht invasive Methode der Thermografie ein Refinement gegenüber anderen Temperaturmessverfahren dar. Diese Methode detektiert Defizite des Ernährungszustandes der Tiere und ermöglicht frühzeitig das Wohlergehen von Nacktmäusen durch geeignete Maßnahmen zu verbessern. Die Thermografie ist ein vielversprechender Ansatz, um die Tierbeurteilung unter anderen Aspekten vorzunehmen, um zukünftig eine bessere tägliche in Augenscheinnahme in der Versuchstierhaltung herbeizuführen. Die qualitative Thermografie stellt sich als geeignete Refinement Methode dar, um die von Nacktmäusen abgestrahlte Wärmestrahlung zu visualisieren und, neben der täglichen Inaugenscheinnahme der Tiere, deren Wohlbefinden objektiv zu beurteilen. Zusätzlich stellt die quantitative Thermografie eine praktische Anwendung dar, um berührungslos und nichtinvasiv die Temperaturen von Nacktmäusen in physiologischen Temperaturbereichen reproduzierbar zu messen. Die Thermografie ist bei Nacktmäusen im Vergleich zu etablierten Temperaturmessverfahren (rektale, subkutane und intraperitoneale) bei gleicher Messgenauigkeit weniger invasiv. Ein Ersatz, der in verschiedenen Studien üblicherweise verwendeten Temperaturerfassung, sollte durch die Möglichkeit, der nicht invasive Methode grundsätzlich abgewogen werden. Die Temperaturmessung mittels Thermografie als nicht invasives Verfahren stellt eine Verbesserung gegenüber etablierten Methoden dar, weil diese nicht mit Belastungen wie die Fixation oder dem Risiko einer Infektion verbunden ist. Im Rahmen von Studien können mit dieser Messmethode im Sinne des 3R - Prinzips nicht manipulativ Temperaturen sicherer an der Nacktmaus gemessen werden.Established methods of measuring body temperature are typically invasive, and may cause mice discomfort and stress. The objective of the study was to compare a non-invasive qualitative thermography with the established invasive temperature measurement methods in nude mice. In this study, the qualitative thermography of an individual and of group-housed nude mice, showed that this method is suitable for measuring the radiated thermal energy (thermal radiation), and for detecting select physiological conditions which result in a change of skin temperature, i.e. bodyweight. An accurate identification using thermal imagery in a grouphoused mouse, is not possible even if additional digital imagery is used for identifying individual animals. Use of qualitative thermograms in group-housed mice is suitable for continuous or repeat visualisation of an individual mouse, as it enables clear differentiation of certain physiological conditions from other animals within that group. The radiated thermal energy of nude mice in the group can be visualised using a qualitative thermogram model infrared camera (IRC) FLIR E40. Quantitative thermography is a practical method for the non-tactile and non-invasive measurement of the temperature of nude mice within physiological ranges. The present study found that a temperature value from a minimum of 10 thermograms should be used for a quantitative measurement. Based on the findings of quantitative thermography, it can be assumed that thermographically measured skin temperatures are not an overestimate. The highest, maximum measured temperature value (irT-max) of all thermograms is the one that most accurately reflects the actual skin temperature. The study results clearly demonstrate that the highest irT-max ever recorded from all thermograms of one quantitative measurement should be used rather than the average of all temperatures recorded, or the average of irTmax of one individual thermogram. The thermographic measurements of 4 female BALB/cAnN-Foxn1nu/nu/Rj mice with different body weights, the measurement of the 50 male Rj:NMRI-Foxn1nu/nu mice and the multiple measurements of the 20 BALB/cAnN-Foxn1nu/nu / Rj male mice over a prolonged period of time demonstrate practical and easy deployment of the thermal imaging. The infrared camera FLIR E40 used for quantitative thermograms has a larger measuring spot size and a lower resolution and quality compared to other infrared cameras that we have evaluated. The high-quality thermal imaging camera FLIR T660 was used to compare the different temperature measurement methods. A smaller size of the measuring spot with infrared camera FLIR T660 made it possible to obtain higher quality irT results as compared to the infrared camera FLIR E40 model. However, automatic focusing is controversial in some areas of research due to the minimal differential between environmental and mouse temperature. This study shows that the auto focus function of the infrared camera FLIR T660 model requires the use of a tripod to prevent vibrations caused by the user, and to ensure high-quality temperature results. Use of a tripod enables focus in a constant plane to generate accurate thermographic measurement in nude mice. The high recording frequency of infrared camera FLIR T660 model, enables radiometric autofocused video recordings, even when the mouse is moving. Thermography is a non-invasive method compared to established temperature measurement methods. The thermal imaging camera is a stress-free measuring device, avoiding surgical intervention, or physical restraint and placement of a foreign object in mice. In our study we have compared IRC with other more invasive methods, the intra-rectal temperature probe, subcutaneous temperature transponders and intra-peritoneal data loggers. Intra-rectal temperature measurements with model 5885, Precision Digital Thermometer with PRT [platinum resistance thermometer] (H Tinsley, New Addington, United Kingdom) probe should be interpreted carefully, as the handling during insertion of the temperature probe tip may result in complications, or modulated response due to stress. Subcutaneous temperature transponders using IPTT-100 (Bio Medic Data Systems, Seaford, DE) also enable individual identification and are useful for some experiments, but transponders must be temperature calibrated. Intra-peritoneal data loggers we used should have the advantage of providing continuous temperature measurement, however with DST Nano–T Temperature Recorder (Starr Oddi, Gardabaer, Iceland) the results of software analysis can only be extracted at the end of experiment. The comparative study results demonstrate that the thermal imager is as accurate as the intrarectal and subcutaneous temperature measurement methods in nude mice. The infrared skin temperature is a valid and accurate method, comparable to rectal and subcutaneous temperature measurements. In summary, the highly efficient, non-invasive method of infra-red thermography is a refinement compared to other more invasive temperature measurement methods. The method can detect deficit in body condition, i.e. bodyweight of nude mice. Qualitative infrared thermography is a suitable refinement method which primary aim is the assessment of surface temperature through capture of thermal radiation emitted by nude mice, and secondarily, for qualitative assessment of overall health status and well-being of animals. In addition, quantitative thermography application can measure reproducibly the temperatures of nude mice in physiological temperature ranges without contact and non-invasively. In nude mice, thermography is non-invasive compared to established temperature measurement methods (rectal, subcutaneous, and intraperitoneal) and with the same measurement accuracy. A substitute for the temperature detection commonly used in various studies by the non-invasive method should always be considered. Temperature measurement using thermography, as a non-invasive method, is an improvement over established methods because it is not associated with physical restraint or the risk of physical damage and subsequent infection. In the context of our studies, this measurement method proved to be safe and accurate for temperature and well-being assessment in nude mice

Monitoring of greenhouse gases and aerosols at Svalbard and Birkenes in 2014 - Annual report.

The report summaries the activities and results of the greenhouse gas monitoring at the Zeppelin Observatory situated on Svalbard in Arctic Norway during the period 2001-2014, and the greenhouse gas monitoring and aerosol observations from Birkenes for 2009-2014

Protease-Activation of Fc-Masked Therapeutic Antibodies to Alleviate Off-Tumor Cytotoxicity

The interaction of the Fc region of therapeutic antibodies and antibody-drug conjugates with Fcγ receptors (FcγRs) can lead to unpredictable and severe side effects. Over the last decades several strategies have been developed to overcome this drawback, including extensive Fc- and glycoengineering and antibody isotype switching. However, these approaches result in permanently Fc-silenced antibody derivates which partially or completely lack antibody-mediated effector functions. Nevertheless, for a majority of antibody-based drugs, Fc-mediated effector functions, like antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) as well as complement-dependent cytotoxicity (CDC), represent the most substantial modes of action. We argued that a new strategy combining the beneficial properties of Fc-silencing and controlled activation of effector functions can pave the way to potent antibody therapeutics, reducing the FcγRs-mediated off-target toxicity. We present a novel Fc-tamed antibody format, where the FcγR-binding sites of antibodies are blocked by anti-isotypic masking units, hindering the association of FcγR and complement component 1 (c1q) to the Fc domain. The masking units were genetically fused to trastuzumab, including a protease-addressable peptide-liker. Our Fc-tamed antibodies demonstrated completely abolished interaction to soluble high-affinity Fcγ-Receptor I and c1q. In reporter cell-based ADCC assays, our Fc-tamed antibodies exhibited a 2,700 to 7,100-fold reduction in activation, compared to trastuzumab. Upon demasking by a tumor-associated protease, the Fc-activated antibodies demonstrated restored FcγR-binding, c1q-binding and the ability to induce potent ADCC activation. Furthermore, cell killing assays using donor-derived NK cells were performed to validate the functionality of the Fc-tamed antibody variants. To our knowledge, this approach represents the first non-permanently Fc-silenced antibody, which can be re-activated by a tumor-associated protease, eventually extending the field of novel antibody formats

Monitoring of greenhouse gases and aerosols at Svalbard and Birkenes in 2012 - Annual report

The report summaries the activities and results of the greenhouse gas monitoring at the Zeppelin and observatory situated on Svalbard in Arctic Norway during the period 2001-2012 and the greenhouse gas monitoring and aerosol observations from Birkenes for 2012. The monitoring programme is performed by the NILU – Norwegian Institute for Air Research and funded by the Norwegian Environment Agency and NILU – Norwegian Institute for Air Research

Safety and immune responses after intradermal application of Porcilis PRRS in either the neck or the perianal region.

The objective of the present study was to assess safety and immune responses in gilts after intradermal application of Porcilis® PRRS in two different application sites under field conditions. Forty-four gilts were allocated to one of three groups: Gilts of group 1 (n = 10) served as non-vaccinated controls, gilts of group 2 (n = 17) were vaccinated intradermally in the neck and gilts of group 3 (n = 17) received an intradermal vaccination in the perianal region. Clinical observations, local injection site reactions and histopathologic examination of the injection site were used for safety assessments. Frequency and degree of clinical signs were not significantly different between all three groups. Minor local reactions for both vaccination groups were observed; however, at 6, 7, 8, 9 and 15 days post-vaccination (dpv), the mean injection site reaction score was significantly lower in pigs vaccinated in the perianal region. In histopathologic examination, an extended inflammatory dimension was observed more frequently in pigs vaccinated in the neck. Blood samples were analyzed to quantify the post-vaccination humoral (ELISA and virus neutralization test) and cellular (IFN-γ ELISPOT) immune responses. PRRSV-specific antibodies were present in the serum of all vaccinated animals from 14 dpv onwards, whereas all control pigs remained negative throughout the study. Neutralizing antibody titers were significantly higher in pigs vaccinated in the perianal region at 28 dpv. At 14, 21 and 28 dpv, PRRSV-specific IFN-γ secreting cells were significantly increased in both vaccination groups compared to non-vaccinated gilts. Analysis of mean numbers of PRRSV-specific IFN-γ secreting cells did not result in statistically significant differences between both vaccination groups. The results of this study indicate that the perianal region is a safe alternative application site for intradermal vaccination of gilts with Porcilis PRRS. Furthermore, the intradermal application of Porcilis PRRS induced humoral and cellular immune responses independent of the administration site