Development of rapid phage based detection methods for mycobacteria

MAP is the causative agent of a wasting disease in ruminants and other animals called Johne’s disease. Culture of the organism can take months and in the case of some sheep strains of MAP, culture can take up to a year. It can take several years for an animal infected with MAP to show clinical symptoms of disease. During this subclinical stage of infection, MAP can be shed into the environment contaminating their surroundings and infecting other animals. As well as this Johne’s disease is particularly difficult to diagnose during the subclinical stage of infection. Culture is very difficult and takes too long to be a viable method to diagnose Johne’s disease. Microscopic methods can be used on histological samples to detect MAP, however common acid-fast stains used are not specific for MAP and other mycobacteria and acid-fast organisms can be detected. Molecular methods, such as PCR, exist to rapidly detect the signature DNA sequences of these organisms, however they have the disadvantage of not being able to distinguish between live and dead organisms. Other immunological methods, such as ELISA tests, exist and are routinely used to diagnose Johne’s disease, however their sensitivity is very poor especially during the subclinical stage of disease. The aim of these studies was to develop novel rapid methods of detecting MAP to act as an alternative to methods already available. Sample processing using magnetic separation was carried out to allow good capture of MAP cells and to allow efficient phage infection. Using the phage assay, a specific, sensitive phage based method was developed that could detect approximately 10 cells per ml of blood within 24 h in the laboratory with a sensitive, specific plaque-PCR. This optimised detection method was then used to determine whether MAP cells could be detected in clinical blood samples of cattle suffering from Johne’s disease. The results suggest that animals experimentally and naturally infected with MAP harboured cells in their blood during subclinical and clinical stages of infection. A novel high-throughput method of detecting mycobacteria was also developed. Using phage D29 as a novel mycobacterial DNA extraction tool, viable MAP cells were detected within 8 h and the format of the assay means that it can be adapted to be used in a high-throughput capacity. Factors affecting phage infection and phage-host interactions were investigated to make sure the phage based methods of detection were as efficient as possible. It was found that periods of recovery were often necessary to not only make sure the phage were not inhibited but to also allow the host cells to be metabolically active as it was found that phage D29 can only infect mycobacteria cells that are metabolically active. A fluorescent fusion-peptide capable of specifically labelling MAP cells was also developed to be used as an alternative to acid-fast staining. Peptides that were found to specifically bind to MAP cells were fused with green fluorescent protein and cells mounted on slides were specifically labelled with the fluorescent fusion protein. This resulted in a good alternative to the generic acid-fast staining methods. The blood phage assay has shown that viable MAP cells can be found in the blood of animals suffering from Johne’s disease within 24 h and this can be confirmed using a MAP specific plaque-PCR protocol. A novel faster method to detect MAP was also developed, to cut down the time to detection of viable MAP cells to 8 h, which can be formatted to be used in a high-throughput capacity. The phage assay was used as a tool to determine different metabolic states of mycobacteria, and helped investigate optimal detection conditions when using the phage assay. Finally a novel fluorescent label was developed to detect MAP as an alternative to insensitive acid-fast staining. The development of these novel methods to rapidly, specifically and sensitively detect MAP will push further the understanding of Johne’s disease and help control it

Development of rapid phage based detection methods for mycobacteria

MAP is the causative agent of a wasting disease in ruminants and other animals called Johne’s disease. Culture of the organism can take months and in the case of some sheep strains of MAP, culture can take up to a year. It can take several years for an animal infected with MAP to show clinical symptoms of disease. During this subclinical stage of infection, MAP can be shed into the environment contaminating their surroundings and infecting other animals. As well as this Johne’s disease is particularly difficult to diagnose during the subclinical stage of infection. Culture is very difficult and takes too long to be a viable method to diagnose Johne’s disease. Microscopic methods can be used on histological samples to detect MAP, however common acid-fast stains used are not specific for MAP and other mycobacteria and acid-fast organisms can be detected. Molecular methods, such as PCR, exist to rapidly detect the signature DNA sequences of these organisms, however they have the disadvantage of not being able to distinguish between live and dead organisms. Other immunological methods, such as ELISA tests, exist and are routinely used to diagnose Johne’s disease, however their sensitivity is very poor especially during the subclinical stage of disease.\ud The aim of these studies was to develop novel rapid methods of detecting MAP to act as an alternative to methods already available. Sample processing using magnetic separation was carried out to allow good capture of MAP cells and to allow efficient phage infection. Using the phage assay, a specific, sensitive phage based method was developed that could detect approximately 10 cells per ml of blood within 24 h in the laboratory with a sensitive, specific plaque-PCR. This optimised detection method was then used to determine whether MAP cells could be detected in clinical blood samples of cattle suffering from Johne’s disease. The results suggest that animals experimentally and naturally infected with MAP harboured cells in their blood during subclinical and clinical stages of infection. A novel high-throughput method of detecting mycobacteria was also developed. Using phage D29 as a novel mycobacterial DNA extraction tool, viable MAP cells were detected within 8 h and the format of the assay means that it can be adapted to be used in a high-throughput capacity. Factors affecting phage infection and phage-host interactions were investigated to make sure the phage based methods of detection were as efficient as possible. It was found that periods of recovery were often necessary to not only make sure the phage were not inhibited but to also allow the host cells to be metabolically active as it was found that phage D29 can only infect mycobacteria cells that are metabolically active. A fluorescent fusion-peptide capable of specifically labelling MAP cells was also developed to be used as an alternative to acid-fast staining. Peptides that were found to specifically bind to MAP cells were fused with green fluorescent protein and cells mounted on slides were specifically labelled with the fluorescent fusion protein. This resulted in a good alternative to the generic acid-fast staining methods. The blood phage assay has shown that viable MAP cells can be found in the blood of animals suffering from Johne’s disease within 24 h and this can be confirmed using a MAP specific plaque-PCR protocol. A novel faster method to detect MAP was also developed, to cut down the time to detection of viable MAP cells to 8 h, which can be formatted to be used in a high-throughput capacity. The phage assay was used as a tool to determine different metabolic states of mycobacteria, and helped investigate optimal detection conditions when using the phage assay. Finally a novel fluorescent label was developed to detect MAP as an alternative to insensitive acid-fast staining. The development of these novel methods to rapidly, specifically and sensitively detect MAP will push further the understanding of Johne’s disease and help control it

Effect of Aqueous Leaf Extract of Jatropha tanjorensis on parasitaemia and haematological parameters in mice infected with Plasmodium ber ghei

Medicinal plants are proven sources of many useful drugs in our modern world. Jatropha tanjorensis, known as hospital too far, is consumed highly in Africa as herbal medicine. This research work was done to evaluate the effect of aqueous leaf extract of Jatropha tanjorensis on parasitaemia and haematological parameters in mice infected with Plasmodium ber ghei. The leaves of J. tanjorensis were extracted with distilled water and qualitative phytochemical analysis carried out. Acute toxicity studies were carried out using the Organization for Economic Cooperation and Development (OECD) guideline. The curative activity of the extract was examined using Rane’s test. A total of forty (40) mice were used for the study and they were infected with Plasmodium berghei. The infected mice were subdivided into five groups of six mice each and treated with different doses of standard drug (artemether/lumefantrine 25mg/kg) and extract (200, 400 and 800mg/kg body weight) for 4 days. After treatment, blood was collected and used for percentage parasitaemia, packed cell volume and hemoglobin concentration. The result of phytochemical analysis revealed the presence of alkaloids, tannins, saponins, flavonoids, terpe noids, cardiac glycosides and anthra quinones. The result of the acute toxicity showed the signs of neither neurological, behavioral nor mortality at concentrations of 2,000 and 5,000 mg/kg oral doses within the first 24 hours and during the 14 days study period. A dose- dependent increase was observed in Total haemoglobin (Hb) and Packed cell volume (PCV) levels which was collaborated with increase in weight as compared with negative control. The leaf extract of J. tanjorensis also revealed a significant (p<0.05) suppression activity and mean survival time at the doses of 400 mg/kg, 200 mg/kg and 100 mg/kg when compared with the negative control. The extract of Jatropha tanjorensis showed reasonable levels of anti-anaemia and antimalarial activities with no signs of acute toxicity.  Therefore this study may support its use as an anti-prophylactic and blood tonic nutraceutical

Scarred Patterns in Surface Waves

Surface wave patterns are investigated experimentally in a system geometry that has become a paradigm of quantum chaos: the stadium billiard. Linear waves in bounded geometries for which classical ray trajectories are chaotic are known to give rise to scarred patterns. Here, we utilize parametrically forced surface waves (Faraday waves), which become progressively nonlinear beyond the wave instability threshold, to investigate the subtle interplay between boundaries and nonlinearity. Only a subset (three main types) of the computed linear modes of the stadium are observed in a systematic scan. These correspond to modes in which the wave amplitudes are strongly enhanced along paths corresponding to certain periodic ray orbits. Many other modes are found to be suppressed, in general agreement with a prediction by Agam and Altshuler based on boundary dissipation and the Lyapunov exponent of the associated orbit. Spatially asymmetric or disordered (but time-independent) patterns are also found even near onset. As the driving acceleration is increased, the time-independent scarred patterns persist, but in some cases transitions between modes are noted. The onset of spatiotemporal chaos at higher forcing amplitude often involves a nonperiodic oscillation between spatially ordered and disordered states. We characterize this phenomenon using the concept of pattern entropy. The rate of change of the patterns is found to be reduced as the state passes temporarily near the ordered configurations of lower entropy. We also report complex but highly symmetric (time-independent) patterns far above onset in the regime that is normally chaotic.Comment: 9 pages, 10 figures (low resolution gif files). Updated and added references and text. For high resolution images: http://physics.clarku.edu/~akudrolli/stadium.htm

Exercise on referral – symposium hosted by the Physical Activity Special Interest Group for the Wolfson Research Institute for Health and Wellbeing, Durham University

The article discusses a symposium on exercise referral schemes (ERS) that was held at the College of St Hild and St Bede, Durham on October 14, 2016. It mentions that exercise referral schemes have increased in popularity to address society's significant chronic disease burden. It also presents the views of Emily Oliver, from Physical Activity Special Interest Group, regarding the same

A concept for application of integrated digital technologies to enhance future smart agricultural systems

Future agricultural systems should increase productivity and sustainability of food production and supply. For this, integrated and efficient capture, management, sharing, and use of agricultural and environmental data from multiple sources is essential. However, there are challenges to understand and efficiently use different types of agricultural and environmental data from multiple sources, which differ in format and time interval. In this regard, the role of emerging technologies is considered to be significant for integrated data gathering, analyses and efficient use. In this study, a concept was developed to facilitate the full integration of digital technologies to enhance future smart and sustainable agricultural systems. The concept has been developed based on the results of a literature review and diverse experiences and expertise which enabled the identification of stat-of-the-art smart technologies, challenges and knowledge gaps. The features of the proposed solution include: data collection methodologies using smart digital tools; platforms for data handling and sharing; application of Artificial Intelligent for data integration and analysis; edge and cloud computing; application of Blockchain, decision support system; and a governance and data security system. The study identified the potential positive implications i.e. the implementation of the concept could increase data value, farm productivity, effectiveness in monitoring of farm operations and decision making, and provide innovative farm business models. The concept could contribute to an overall increase in the competitiveness, sustainability, and resilience of the agricultural sector as well as digital transformation in agriculture and rural areas. This study also provided future research direction in relation to the proposed concept. The results will benefit researchers, practitioners, developers of smart tools, and policy makers supporting the transition to smarter and more sustainable agriculture systems

IMpower030: Phase III study evaluating neoadjuvant treatment of resectable stage II-IIIB non-small cell lung cancer (NSCLC) with atezolizumab (atezo) + chemotherapy

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Immunosenescence (iSenescence) correlates with progression (PD) to PD-(L)1 inhibitors (IO) and not to platinum-chemotherapy (PCT) in advanced non-small cell lung cancer (aNSCLC) patients (pts)

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Survival of Mycobacterium avium subspecies paratuberculosis in retail pasteurised milk

A survey of retail purchased semi-skimmed pasteurised milk (n = 368) for Mycobacterium avium subspecies paratuberculosis (MAP) was conducted between May 2014 and June 2015 across the midlands of England using the Phage-PCR assay. Overall, 10.3% of the total samples collected contained viable MAP cells, confirming that pasteurisation is not capable of fully eliminating human exposure to viable MAP through milk. Comparison of the results gained using the Phage-PCR assay with the results of surveys using either culture or direct PCR suggest that the phage-PCR assay is able to detect lower numbers of cells, resulting in an increase in the number of MAP-positive samples detected. Comparison of viable count and levels of MAP detected in bulk milk samples suggest that MAP is not primarily introduced into the milk by faecal contamination but rather are shed directly into the milk within the udder. In addition results detected an asymmetric distribution of MAP exists in the milk matrix prior to somatic cell lysis, indicating that the bacterial cells in naturally contaminated milk are clustered together and may primarily be located within somatic cells. These latter two results lead to the hypothesis that intracellular MAP within the somatic cells may be protected against heat inactivation during pasteurisation, accounting for the presence of low levels of MAP detected in retail milk

Study of Effect on Teeth of Intermittent Fluoridation of a Community Water Supply

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/67913/2/10.1177_00220345530320011601.pd