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

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

Measurement of the cross section for isolated-photon plus jet production in pp collisions at √s=13 TeV using the ATLAS detector

The dynamics of isolated-photon production in association with a jet in proton–proton collisions at a centre-of-mass energy of 13 TeV are studied with the ATLAS detector at the LHC using a dataset with an integrated luminosity of 3.2 fb−1. Photons are required to have transverse energies above 125 GeV. Jets are identified using the anti- algorithm with radius parameter and required to have transverse momenta above 100 GeV. Measurements of isolated-photon plus jet cross sections are presented as functions of the leading-photon transverse energy, the leading-jet transverse momentum, the azimuthal angular separation between the photon and the jet, the photon–jet invariant mass and the scattering angle in the photon–jet centre-of-mass system. Tree-level plus parton-shower predictions from Sherpa and Pythia as well as next-to-leading-order QCD predictions from Jetphox and Sherpa are compared to the measurements

A search for resonances decaying into a Higgs boson and a new particle X in the XH → qqbb final state with the ATLAS detector

A search for heavy resonances decaying into a Higgs boson (H) and a new particle (X) is reported, utilizing 36.1 fb−1 of proton–proton collision data at collected during 2015 and 2016 with the ATLAS detector at the CERN Large Hadron Collider. The particle X is assumed to decay to a pair of light quarks, and the fully hadronic final state is analysed. The search considers the regime of high XH resonance masses, where the X and H bosons are both highly Lorentz-boosted and are each reconstructed using a single jet with large radius parameter. A two-dimensional phase space of XH mass versus X mass is scanned for evidence of a signal, over a range of XH resonance mass values between 1 TeV and 4 TeV, and for X particles with masses from 50 GeV to 1000 GeV. All search results are consistent with the expectations for the background due to Standard Model processes, and 95% CL upper limits are set, as a function of XH and X masses, on the production cross-section of the resonance

Combination of searches for Higgs boson pairs in pp collisions at \sqrts = 13 TeV with the ATLAS detector

This letter presents a combination of searches for Higgs boson pair production using up to 36.1 fb(-1) of proton-proton collision data at a centre-of-mass energy root s = 13 TeV recorded with the ATLAS detector at the LHC. The combination is performed using six analyses searching for Higgs boson pairs decaying into the b (b) over barb (b) over bar, b (b) over barW(+)W(-), b (b) over bar tau(+)tau(-), W+W-W+W-, b (b) over bar gamma gamma and W+W-gamma gamma final states. Results are presented for non-resonant and resonant Higgs boson pair production modes. No statistically significant excess in data above the Standard Model predictions is found. The combined observed (expected) limit at 95% confidence level on the non-resonant Higgs boson pair production cross-section is 6.9 (10) times the predicted Standard Model cross-section. Limits are also set on the ratio (kappa(lambda)) of the Higgs boson self-coupling to its Standard Model value. This ratio is constrained at 95% confidence level in observation (expectation) to -5.0 &lt; kappa(lambda) &lt; 12.0 (-5.8 &lt; kappa(lambda) &lt; 12.0). In addition, limits are set on the production of narrow scalar resonances and spin-2 Kaluza-Klein Randall-Sundrum gravitons. Exclusion regions are also provided in the parameter space of the habemus Minimal Supersymmetric Standard Model and the Electroweak Singlet Model. For complete list of authors see http://dx.doi.org/10.1016/j.physletb.2019.135103</p

Searches for lepton-flavour-violating decays of the Higgs boson in s=13\sqrt{s}=13 TeV pp\mathit{pp} collisions with the ATLAS detector

This Letter presents direct searches for lepton flavour violation in Higgs boson decays, H → eτ and H → μτ , performed with the ATLAS detector at the LHC. The searches are based on a data sample of proton–proton collisions at a centre-of-mass energy √s = 13 TeV, corresponding to an integrated luminosity of 36.1 fb−1. No significant excess is observed above the expected background from Standard Model processes. The observed (median expected) 95% confidence-level upper limits on the leptonflavour-violating branching ratios are 0.47% (0.34+0.13−0.10%) and 0.28% (0.37+0.14−0.10%) for H → eτ and H → μτ , respectively.publishedVersio

Comparison between simulated and observed LHC beam backgrounds in the ATLAS experiment at Ebeam =4 TeV

Results of dedicated Monte Carlo simulations of beam-induced background (BIB) in the ATLAS experiment at the Large Hadron Collider (LHC) are presented and compared with data recorded in 2012. During normal physics operation this background arises mainly from scattering of the 4 TeV protons on residual gas in the beam pipe. Methods of reconstructing the BIB signals in the ATLAS detector, developed and implemented in the simulation chain based on the \textscFluka Monte Carlo simulation package, are described. The interaction rates are determined from the residual gas pressure distribution in the LHC ring in order to set an absolute scale on the predicted rates of BIB so that they can be compared quantitatively with data. Through these comparisons the origins of the BIB leading to different observables in the ATLAS detectors are analysed. The level of agreement between simulation results and BIB measurements by ATLAS in 2012 demonstrates that a good understanding of the origin of BIB has been reached

Search for an invisibly decaying Higgs boson or dark matter candidates produced in association with a Z boson in pp collisions at root s=13 TeV with the ATLAS detector

SCOAP

Search for flavour-changing neutral currents in processes with one top quark and a photon using 81 fb⁻¹ of pp collisions at \sqrts = 13 TeV with the ATLAS experiment

A search for flavour-changing neutral current (FCNC) events via the coupling of a top quark, a photon, and an up or charm quark is presented using 81 fb−1 of proton–proton collision data taken at a centre-of-mass energy of 13 TeV with the ATLAS detector at the LHC. Events with a photon, an electron or muon, a b-tagged jet, and missing transverse momentum are selected. A neural network based on kinematic variables differentiates between events from signal and background processes. The data are consistent with the background-only hypothesis, and limits are set on the strength of the tqγ coupling in an effective field theory. These are also interpreted as 95% CL upper limits on the cross section for FCNC tγ production via a left-handed (right-handed) tuγ coupling of 36 fb (78 fb) and on the branching ratio for t→γu of 2.8×10−5 (6.1×10−5). In addition, they are interpreted as 95% CL upper limits on the cross section for FCNC tγ production via a left-handed (right-handed) tcγ coupling of 40 fb (33 fb) and on the branching ratio for t→γc of 22×10−5 (18×10−5). © 2019 The Author(s