A molecular epidemiological investigation of contagious caprine pleuropneumonia in goats and captive Arabian sand gazelle (Gazella marica) in Oman.

BACKGROUND: Contagious caprine pleuropneumonia (CCPP) is a fatal WOAH-listed, respiratory disease in small ruminants with goats as primary hosts that is caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp). Twelve CCPP outbreaks were investigated in 11 goat herds and a herd of captive Arabian sand gazelle (Gazella marica) in four Omani governorates by clinical pathological and molecular analysis to compare disease manifestation and Mccp genetic profiles in goats and wild ungulates. RESULTS: The CCPP forms in diseased and necropsied goats varied from peracute (5.8%), acute (79.2%) and chronic (4.5%) while all of the five necropsied gazelles showed the acute form based on the clinical picture, gross and histopathological evaluation. Colonies of Mccp were recovered from cultured pleural fluid, but not from lung tissue samples of one gazelle and nine goats and all the isolates were confirmed by Mccp-specific real time PCR. Whole genome-single nucleotide polymorphism (SNP) analysis was performed on the ten isolates sequenced in this study and twenty sequences retrieved from the Genbank database. The Mccp strains from Oman clustered all in phylogroup A together with strains from East Africa and one strain from Qatar. A low variability of around 125 SNPs was seen in the investigated Omani isolates from both goats and gazelles indicating mutual transmission of the pathogen between wildlife and goats. CONCLUSION: Recent outbreaks of CCPP in Northern Oman are caused by Mccp strains of the East African Phylogroup A which can infect goats and captive gazelles likewise. Therefore, wild and captive ungulates should be considered as reservoirs and included in CCPP surveillance measures.

Minimalistic mycoplasmas harbor different functional toxin-antitoxin systems.

Mycoplasmas are minute bacteria controlled by very small genomes ranging from 0.6 to 1.4 Mbp. They encompass several important medical and veterinary pathogens that are often associated with a wide range of chronic diseases. The long persistence of mycoplasma cells in their hosts can exacerbate the spread of antimicrobial resistance observed for many species. However, the nature of the virulence factors driving this phenomenon in mycoplasmas is still unclear. Toxin-antitoxin systems (TA systems) are genetic elements widespread in many bacteria that were historically associated with bacterial persistence. Their presence on mycoplasma genomes has never been carefully assessed, especially for pathogenic species. Here we investigated three candidate TA systems in M. mycoides subsp. capri encoding a (i) novel AAA-ATPase/subtilisin-like serine protease module, (ii) a putative AbiEii/AbiEi pair and (iii) a putative Fic/RelB pair. We sequence analyzed fourteen genomes of M. mycoides subsp. capri and confirmed the presence of at least one TA module in each of them. Interestingly, horizontal gene transfer signatures were also found in several genomic loci containing TA systems for several mycoplasma species. Transcriptomic and proteomic data confirmed differential expression profiles of these TA systems during mycoplasma growth in vitro. While the use of heterologous expression systems based on E. coli and B. subtilis showed clear limitations, the functionality and neutralization capacities of all three candidate TA systems were successfully confirmed using M. capricolum subsp. capricolum as a host. Additionally, M. capricolum subsp. capricolum was used to confirm the presence of functional TA system homologs in mycoplasmas of the Hominis and Pneumoniae phylogenetic groups. Finally, we showed that several of these M. mycoides subsp. capri toxins tested in this study, and particularly the subtilisin-like serine protease, could be used to establish a kill switch in mycoplasmas for industrial applications.

De novo genome assembly resolving repetitive structures enables genomic analysis of 35 European Mycoplasmopsis bovis strains.

Mycoplasmopsis (M.) bovis, the agent of mastitis, pneumonia, and arthritis in cattle, harbors a small genome of approximately 1 Mbp. Combining data from Illumina and Nanopore technologies, we sequenced and assembled the genomes of 35 European strains and isolate DL422_88 from Cuba. While the high proportion of repetitive structures in M. bovis genomes represent a particular challenge, implementation of our own pipeline Mycovista (available on GitHub www.github.com/sandraTriebel/mycovista ) in a hybrid approach enabled contiguous assembly of the genomes and, consequently, improved annotation rates considerably. To put our European strain panel in a global context, we analyzed the new genome sequences together with 175 genome assemblies from public databases. Construction of a phylogenetic tree based on core genes of these 219 strains revealed a clustering pattern according to geographical origin, with European isolates positioned on clades 4 and 5. Genomic data allowing assignment of strains to tissue specificity or certain disease manifestations could not be identified. Seven strains isolated from cattle with systemic circular condition (SCC), still a largely unknown manifestation of M. bovis disease, were located on both clades 4 and 5. Pairwise association analysis revealed 108 genomic elements associated with a particular clade of the phylogenetic tree. Further analyzing these hits, 25 genes are functionally annotated and could be linked to a M. bovis protein, e.g. various proteases and nucleases, as well as ten variable surface lipoproteins (Vsps) and other surface proteins. These clade-specific genes could serve as useful markers in epidemiological and clinical surveys.

Do diets with higher carbon footprints increase the risk of mortality? A population-based simulation study using self-selected diets from the USA.

OBJECTIVE: Are diets with a greater environmental impact less healthy? This is a key question for nutrition policy, but previous research does not provide a clear answer. To address this, our objective here was to test whether American diets with the highest carbon footprints predicted greater population-level mortality from diet-related chronic disease than those with the lowest. DESIGN: Baseline dietary recall data were combined with a database of greenhouse gases emitted in the production of foods to estimate a carbon footprint for each diet. Diets were ranked on their carbon footprints and those in the highest and lowest quintiles were studied here. Preventable Risk Integrated Model (PRIME), an epidemiological modelling software, was used to assess CVD and cancer mortality for a simulated dietary change from the highest to the lowest impact diets. The diet-mortality relationships used by PRIME came from published meta-analyses of randomised controlled trials and prospective cohort studies. SETTING: USA. PARTICIPANTS: Baseline diets came from adults (n 12 865) in the nationally representative 2005-2010 National Health and Nutrition Examination Survey. RESULTS: A simulated change at the population level from the highest to the lowest carbon footprint diets resulted in 23 739 (95 % CI 20 349, 27 065) fewer annual deaths from CVD and cancer. This represents a 1·83 % (95 % CI 1·57 %, 2·08 %) decrease in total deaths. About 95 % of deaths averted were from CVD. CONCLUSIONS: Diets with the highest carbon footprints were associated with a greater risk of mortality than the lowest, suggesting that dietary guidance could incorporate sustainability information to reinforce health messaging.

The carbon footprint of dietary guidelines around the world: a seven country modeling study.

BACKGROUND: Do the environmental impacts inherent in national food-based dietary guidelines (FBDG) vary around the world, and, if so, how? Most previous studies that consider this question focus on a single country or compare countries' guidelines without controlling for differences in country-level consumption patterns. To address this gap, we model the carbon footprint of the dietary guidelines from seven different countries, examine the key contributors to this, and control for consumption differences between countries. METHODS: In this purposive sample, we obtained FBDG from national sources for Germany, India, the Netherlands, Oman, Thailand, Uruguay, and the United States. These were used to structure recommended diets using 6 food groups: protein foods, dairy, grains, fruits, vegetables, and oils/fats. To determine specific quantities of individual foods within these groups, we used data on food supplies available for human consumption for each country from the UN Food and Agriculture Organization's food balance sheets. The greenhouse gas emissions (GHGE) used to produce the foods in these consumption patterns were linked from our own database, constructed from an exhaustive review of the life cycle assessment literature. All guidelines were scaled to a 2000-kcal diet. RESULTS: Daily recommended amounts of dairy foods ranged from a low of 118 ml/d for Oman to a high of 710 ml/d for the US. The GHGE associated with these two recommendations were 0.17 and 1.10 kg CO2-eq/d, respectively. The GHGE associated with the protein food recommendations ranged from 0.03 kg CO2-eq/d in India  to 1.84 kg CO2-eq/d in the US, for recommended amounts of 75 g/d and 156 g/d, respectively. Overall, US recommendations had the highest carbon footprint at 3.83 kg CO2-eq/d, 4.5 times that of the recommended diet for India, which had the smallest footprint. After controlling for country-level consumption patterns by applying the US consumption pattern to all countries, US recommendations were still the highest, 19% and 47% higher than those of the Netherlands and Germany, respectively. CONCLUSIONS: Despite our common human biology, FBDG vary tremendously from one country to the next, as do the associated carbon footprints of these guidelines. Understanding the carbon footprints of different recommendations can assist in future decision-making to incorporate environmental sustainability in dietary guidance.

Biofunctionalization of porcine-derived collagen matrices with platelet rich fibrin: influence on angiogenesis in vitro and in vivo.

OBJECTIVES: Porcine-derived collagen matrices (CM) can be used for oral tissue regeneration, but sufficient revascularization is crucial. The aim of this study was to analyze the influence of platelet-rich fibrin (PRF) on angiogenesis of different CM in vitro and in vivo. MATERIALS AND METHODS: Three different CM (mucoderm®, jason®, collprotect®) were combined with PRF in a plotting process. Growth factor release (VEGF, TGF-ß) was measured in vitro via ELISA quantification after 1,4 and 7 days in comparison to PRF alone. In ovo yolk sac (YSM) and chorion allantois membrane (CAM) model, angiogenic potential were analyzed in vivo with light- and intravital fluorescence microscopy after 24 h, then verified with immunohistochemical staining for CD105 and αSMA. RESULTS: Highest growth factor release was seen after 24 h for all three activated membranes in comparison to the native CM (VEGF 24 h: each p < 0.05; TGF-ß: each p < 0.001) and the PRF (no significant difference). All activated membranes revealed a significantly increased angiogenic potential in vivo after 24 h (vessels per mm2: each p < 0.05; branching points per mm2: each p < 0.01; vessel density: each p < 0.05) and with immunohistochemical staining for CD105 (each p < 0.01) and αSMA (each p < 0.05). CONCLUSIONS: PRF improved the angiogenesis of CM in vitro and in vivo. CLINICAL RELEVANCE: Bio-functionalization of CM with PRF could easily implemented in the clinical pathway and may lead to advanced soft tissue healing.

The Impact of a Real-Time Locating System within the Perioperative Environment on Physicians and Patients' Families.

BACKGROUND: Humber River Hospital has implemented a real-time location system (RTLS) within the operating room in order to provide real-time information about patients' status and manage the many components involved during the perioperative journey. OBJECTIVE: The aim of this study was to explore both physicians' and family members' perceptions of the functionality and efficiency of the RTLS within the perioperative environment. METHODS: Semi-structured interviews were conducted with physicians and patients' family members to elicit various perspectives regarding the use of RTLSs throughout the perioperative process. Interviews were recorded and transcribed to extract key themes. RESULTS: Three themes gleaned from physician interviews were system weaknesses, perceptions of potential benefit, and benefits to family members. Three themes uncovered from family member interviews included convenience, ameliorating anxiety, and reducing interruptions. CONCLUSION: Overall, physicians reported that the RTLS had potential to enhance workflow but that significant improvement regarding its implementation and use was needed to reach its full benefit. Family members were unanimous that it provides them with all the tracking information they desire.

Reproduction of contagious caprine pleuropneumonia reveals the ability of convalescent sera to reduce hydrogen peroxide production in vitro.

Contagious caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subsp. capripneumoniae is a severe disease widespread in Africa and Asia. Limited knowledge is available on the pathogenesis of this organism, mainly due to the lack of a robust in vivo challenge model and the means to do site-directed mutagenesis. This work describes the establishment of a novel caprine challenge model for CCPP that resulted in 100% morbidity using a combination of repeated intranasal spray infection followed by a single transtracheal infection employing the recent Kenyan outbreak strain ILRI181. Diseased animals displayed CCPP-related pathology and the bacteria could subsequently be isolated from pleural exudates and lung tissues in concentrations of up to 109 bacteria per mL as well as in the trachea using immunohistochemistry. Reannotation of the genome sequence of ILRI181 and F38T revealed the existence of genes encoding the complete glycerol uptake and metabolic pathways involved in hydrogen peroxide (H2O2) production in the phylogenetically related pathogen M. mycoides subsp. mycoides. Furthermore, the expression of L-α-glycerophosphate oxidase (GlpO) in vivo was confirmed. In addition, the function of the glycerol metabolism was verified by measurement of production of H2O2 in medium containing physiological serum concentrations of glycerol. Peroxide production could be inhibited with serum from convalescent animals. These results will pave the way for a better understanding of host-pathogen interactions during CCPP and subsequent vaccine development.

Milestone prediction for time-to-event endpoint monitoring in clinical trials.

Predicting the times of milestone events, ie, interim and final analyses in clinical trials, helps resource planning. This manuscript presents and compares several easily implemented methods for predicting when a milestone event is achieved. We show that it is beneficial to combine the predictions from different models to craft a better predictor through prediction synthesis. Furthermore, a Bayesian approach provides a better measure of the uncertainty involved in prediction of milestone events. We compare the methods through two simulations where the model has been correctly specified and where the models are a mixture of three incorrectly specified model classes. We then apply the methods on two real clinical trial data, North Central Cancer Treatment Group (NCCTG) N0147 and N9841. In summary, the Bayesian prediction synthesis methods automatically perform well even when the data collection is far from homogeneous. An R shiny app is under development to carry out the prediction in a user-friendly fashion.

Cities' Role in Mitigating United States Food System Greenhouse Gas Emissions.

Current trends of urbanization, population growth, and economic development have made cities a focal point for mitigating global greenhouse gas (GHG) emissions. The substantial contribution of food consumption to climate change necessitates urban action to reduce the carbon intensity of the food system. While food system GHG mitigation strategies often focus on production, we argue that urban influence dominates this sector's emissions and that consumers in cities must be the primary drivers of mitigation. We quantify life cycle GHG emissions of the United States food system through data collected from literature and government sources producing an estimated total of 3800 kg CO2e/capita in 2010, with cities directly influencing approximately two-thirds of food sector GHG emissions. We then assess the potential for cities to reduce emissions through selected measures; examples include up-scaling urban agriculture and home delivery of grocery options, which each may achieve emissions reductions on the order of 0.4 and ∼1% of this total, respectively. Meanwhile, changes in waste management practices and reduction of postdistribution food waste by 50% reduce total food sector emissions by 5 and 11%, respectively. Consideration of the scale of benefits achievable through policy goals can enable cities to formulate strategies that will assist in achieving deep long-term GHG emissions targets.

Spatial Variability and Uncertainty of Water Use Impacts from U.S. Feed and Milk Production.

This paper addresses water use impacts of agriculture, developing a spatially explicit approach tracing the location of water use and water scarcity related to feed production, transport, and livestock, tracking uncertainties and illustrating the approach with a case study on dairy production in the United States. This approach was developed as a step to bring spatially variable production and impacts into a process-based life cycle assessment (LCA) context. As water resources and demands are spatially variable, it is critical to take into account the location of activities to properly understand the impacts of water use, accounting for each of the main feeds for milk production. At the crop production level, the example of corn grain shows that 59% of water stress associated with corn grain production in the United States is located in Nebraska, a state with moderate water stress and moderate corn production (11%). At the level of milk production, four watersheds account for 78% of the national water stress impact, as these areas have high milk production and relatively high water stress; it is the production of local silage and hay crops that drives water consumption in these areas. By considering uncertainty in both inventory data and impact characterization factors, we demonstrate that spatial variability may be larger than uncertainty, and that not systematically accounting for the two can lead to artificially high uncertainty. Using a nonspatial approach in a spatially variable setting can result in a significant underestimation or overestimation of water impacts. The approach demonstrated here could be applied to other spatially variable processes.

Comparison of growth & function of endothelial progenitor cells cultured on deproteinized bovine bone modified with covalently bound fibronectin and bound vascular endothelial growth factor.

OBJECTIVES: The objective of this study was to assess and compare the growth and function of Endothelial Progenitor Cells (EPCs) cultured on covalently bonded Vascular Endothelial Growth Factor (VEGF) and covalently bonded Fibronectin (FN) coating on deproteinized bovine bone (DBB) (test samples), compared to non-modified DBB blocks (control sample). MATERIALS AND METHODS: The test samples were prepared by plasma polymerization of allylamine onto DBB blocks. Group1 of test samples were prepared with VEGF coating (VEGF-DBB) where as the Group2 test samples were coated with FN (FN-DBB). Non-modified DBB blocks served as a Control. EPCs were isolated and cultivated from buffy coats of peripheral blood of healthy volunteers and cultivated in the different samples and examined at time intervals of 24 h, 3 days, and 7 days. Evaluation of growth by cell count and cell morphology was done using Confocal Laser Scanning Electron Microscopy; vitality and function of cells was assessed using MTT assay and RT-PCR and ELISA for eNOS and iNOS respectively. RESULTS: The results of the study show that both VEGF and FN could be successfully immobilized by plasma polymerization onto a complex, porous, three-dimensional structure of DBB. When comparing vital cell coverage, proliferation and function of EPCs, FN-DBB provided more positive values followed by VEGF-DBB as compared to DBB samples. eNOS level were significant higher in VEGF-DBB and FN-DBB when compared to DBB (P = 0.019 and P = 0.002). The difference between VEGF-DBB and FN-DBB was not significant. CONCLUSIONS: Biomimetic coatings of Fibronectin may clinically relate to faster angiogenesis and earlier healing potential.

Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia.

Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused by Mycoplasma mycoides subsp. mycoides, a bacterium belonging to the Mycoplasma mycoides cluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenic Mycoplasma mycoides subsp. mycoides proteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinant Mycoplasma immunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP.

Tick infestation in birds and prevalence of pathogens in ticks collected from different places in Germany.

The importance of ticks and tick-borne pathogens for human and animal health has been increasing over the past decades. For their transportation and dissemination, birds may play a more important role than wingless hosts. In this study, tick infestation of birds in Germany was examined. Eight hundred ninety-two captured birds were infested with ticks and belonged to 48 different species, of which blackbirds (Turdus merula) and song thrushes (Turdus philomelos) were most strongly infested. Ground feeders were more strongly infested than non-ground feeders, sedentary birds more strongly than migratory birds, and short-distance migratory birds more strongly than long-distance migratory birds. Mean tick infestation per bird ranged between 2 (long-distance migratory bird) and 4.7 (sedentary bird), in some single cases up to 55 ticks per bird were found. With the exception of three nymphs of Haemaphysalis spp., all ticks belonged to Ixodes spp., the most frequently detected tick species was Ixodes ricinus. Birds were mostly infested by nymphs (65.1 %), followed by larvae (32.96 %). Additionally, ticks collected from birds were examined for several pathogens: Tick-borne encephalitis virus (TBEV) and Sindbisvirus with real-time RT-PCR, Flaviviruses, Simbuviruses and Lyssaviruses with broad-range standard RT-PCR-assays, and Borrelia spp. with a Pan-Borrelia real-time PCR. Interestingly, no viral pathogens could be detected, but Borrelia spp. positive ticks were collected from 76 birds. Borrelia (B.) garinii, B. valaisiaina, B. burgdorferi s.s. and B. afzelii were determined. The screening of ticks and birds for viral pathogens with broad range PCR-assays was tested and the use as an "early warning system" is discussed.

Social and legal frame conditions for 3D (and) bioprinting in medicine.

The beginnings of three-dimensional (3D) printing and bioprinting can be traced to as early as 1984. From printing inorganic models for the generation of biologic scaffolds, additive manufacturing (AM) developed to the direct printing of organic materials, including specialized tissues, proteins, and cells. In recent years, these technologies have gained significantly in relevance, and there have been several innovations, especially in the field of regenerative medicine. It is becoming increasingly important to consider the economic and social aspects of AM, particularly in education and information of medical human resources, society, and politics, as well as for the establishment of homogenous, globally adapted legal regulations.

Applications of patient-specific 3D printing in medicine.

Already three decades ago, the potential of medical 3D printing (3DP) or rapid prototyping for improved patient treatment began to be recognized. Since then, more and more medical indications in different surgical disciplines have been improved by using this new technique. Numerous examples have demonstrated the enormous benefit of 3DP in the medical care of patients by, for example, planning complex surgical interventions preoperatively, reducing implantation steps and anesthesia times, and helping with intraoperative orientation. At the beginning of every individual 3D model, patient-specific data on the basis of computed tomography (CT), magnetic resonance imaging (MRI), or ultrasound data is generated, which is then digitalized and processed using computer-aided design/computer-aided manufacturing (CAD/CAM) software. Finally, the resulting data sets are used to generate 3D-printed models or even implants. There are a variety of different application areas in the various medical fields, eg, drill or positioning templates, or surgical guides in maxillofacial surgery, or patient-specific implants in orthopedics. Furthermore, in vascular surgery it is possible to visualize pathologies such as aortic aneurysms so as to improve the planning of surgical treatment. Although rapid prototyping of individual models and implants is already applied very successfully in regenerative medicine, most of the materials used for 3DP are not yet suitable for implantation in the body. Therefore, it will be necessary in future to develop novel therapy approaches and design new materials in order to completely reconstruct natural tissue.

Materials and scaffolds in medical 3D printing and bioprinting in the context of bone regeneration.

The structural and functional repair of lost bone is still one of the biggest challenges in regenerative medicine. In many cases, autologous bone is used for the reconstruction of bone tissue; however, the availability of autologous material is limited, which always means additional stress to the patient. Due to this, more and more frequently various biocompatible materials are being used instead for bone augmentation. In this context, in order to ensure the structural function of the bone, scaffolds are implanted and fixed into the bone defect, depending on the medical indication. Nevertheless, for the surgeon, every individual clinical condition in which standardized scaffolds have to be aligned is challenging, and in many cases the alignment is not possible without limitations. Therefore, in the last decades, 3D printing (3DP) or additive manufacturing (AM) of scaffolds has become one of the most innovative approaches in surgery to individualize and improve the treatment of patients. Numerous biocompatible materials are available for 3DP, and various printing techniques can be applied, depending on the process conditions of these materials. Besides these conventional printing techniques, another promising approach in the context of medical AM is 3D bioprinting, a technique which makes it possible to print human cells embedded in special carrier substances to generate functional tissues. Even the direct printing into bone defects or lesions becomes possible. 3DP is already improving the treatment of patients, and has the potential to revolutionize regenerative medicine in future.

Morphological characterization and immunohistochemical detection of the proinflammatory cytokines IL-1ß, IL-17A, and TNF-α in lung lesions associated with contagious bovine pleuropneumonia.

Contagious bovine pleuropneumonia (CBPP), a severe respiratory disease, is characterized by massive inflammation of the lung especially during the acute clinical stage of infection. Tissue samples from cattle, experimentally infected with Mycoplasma mycoides subsp. mycoides Afadé, were subjected to histopathological and immunohistochemical examination in order to provide insight into innate immune pathways that shape inflammatory host responses. Lung lesions were characterized by vasculitis, necrosis, and increased presence of macrophages and neutrophils, relative to uninfected animals. The presence of three cytokines associated with innate inflammatory immune responses, namely, IL-1ß, IL-17A, and TNF-α, were qualitatively investigated in situ. Higher cytokine levels were detected in lung tissue samples from CBPP-affected cattle compared to samples derived from an uninfected control group. We therefore conclude that the cytokines TNF-α and IL-1ß, which are prevalent in the acute phase of infections, play a role in the inflammatory response seen in the lung tissue in CBPP. IL-17A gets released by activated macrophages and attracts granulocytes that modulate the acute phase of the CBPP lesions.

Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.

Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae that affects goats in Africa and Asia. Current available methods for the diagnosis of Mycoplasma infection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection of M. capricolum subsp. capripneumoniae. We developed the assay using a specific target sequence in M. capricolum subsp. capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 10(3) and 5 × 10(4) cells/ml were obtained using genomic DNA and bacterial culture from M. capricolum subsp. capripneumoniae strain ILRI181, while no amplification was obtained from 71 related Mycoplasma isolates or from the Acholeplasma or the Pasteurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting.

Investigation of medical intervention with fatal outcome: the impact of post-mortem CT and CT angiography.

Post-mortem computed tomography (PMCT) has been proven for its appropriateness to become an integral part of routine pre-autoptic forensic investigations either in the field of forensic investigation of fatal medical error or in hospital quality management. The autoptic investigation of unexpected and peri-interventional deaths can be usefully guided by post-mortem imaging which offers significant added value in the documentation of misplacement of medical devices before dissection with the risk of artificial relocation and the detection of iatrogenic air embolism. Post-mortem CT angiography (PMCTA) augments PMCT in the search for sources of hemorrhages and for the documentation of vascular patency and unimpaired perfusion after general and cardiovascular surgery or transvascular catheter-assisted interventions. Limitations of PMCT and PMCTA in medical error cases are method-related or time-dependent including artifacts by early post-mortem tissue change. Thromboembolic complications including pulmonary embolism, the differentiation of ante- and post-mortem coagulation and the detection of myocardial infarction remain areas with compromised diagnostic efficiency as compared to autopsy. Furthermore, extended survival periods after a complication in question impedes visualization of contrast agent extravasation at vascular leakage sites. PMCT and PMCTA contribute substantially for proving a correct interventional approach and guide forensic or clinical autopsy in the reconstruction of adverse medical events with fatal outcome. Post-mortem imaging could also assume a new role as an alternative in a clinicopathological setting if autopsy is not achievable when the probability in the individual case is acceptable to answer specific questions.