186 research outputs found
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
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Treatment of plutonium contaminated soil/sediment from the Mound site using the ACT*DE*CON{sup SM} process
The removal and/or treatment of contaminated soil is a major problem facing the US DOE. The EG&G Mound Applied Technologies site in Miamisburg, Ohio, has an estimated 1.5 million cubic feet of soils from past disposal and waste burial practices awaiting remediation from plutonium contamination. This amount includes sediment from the Miami-Erie Canal that was contaminated in 1969 following a pipe- rupture accident. Conventional soil washing techniques that use particle separation would generate too large a waste volume to be economically feasible. Therefore, innovative technologies are needed for the cleanup. The ACT*DE*CON process was developed by SELENTEC for washing soils to selectively dissolve and remove heavy metals and radionuclides. ACT*DE*CON chemically dissolves and removes heavy metals and radionuclides from soils and sediments into an aqueous medium. The ACT*DE*CON process uses oxidative carbonate/chelant chemistry to dissolve the contaminant from the sediment and hold the contaminant in solution. The objective of recent work was to document the proves conditions necessary to achieve the Mound-site and regulatory-cleanup goals using the ACT*DE*CON technology
Numerical study of pattern formation following a convective instability in non-Boussinesq fluids
We present a numerical study of a model of pattern formation following a
convective instability in a non-Boussinesq fluid. It is shown that many of the
features observed in convection experiments conducted on gas can be
reproduced by using a generalized two-dimensional Swift-Hohenberg equation. The
formation of hexagonal patterns, rolls and spirals is studied, as well as the
transitions and competition among them. We also study nucleation and growth of
hexagonal patterns and find that the front velocity in this two dimensional
model is consistent with the prediction of marginal stability theory for one
dimensional fronts.Comment: 9 pages, report FSU-SCRI-92-6
Mean flow and spiral defect chaos in Rayleigh-Benard convection
We describe a numerical procedure to construct a modified velocity field that
does not have any mean flow. Using this procedure, we present two results.
Firstly, we show that, in the absence of mean flow, spiral defect chaos
collapses to a stationary pattern comprising textures of stripes with angular
bends. The quenched patterns are characterized by mean wavenumbers that
approach those uniquely selected by focus-type singularities, which, in the
absence of mean flow, lie at the zig-zag instability boundary. The quenched
patterns also have larger correlation lengths and are comprised of rolls with
less curvature. Secondly, we describe how mean flow can contribute to the
commonly observed phenomenon of rolls terminating perpendicularly into lateral
walls. We show that, in the absence of mean flow, rolls begin to terminate into
lateral walls at an oblique angle. This obliqueness increases with Rayleigh
number.Comment: 14 pages, 19 figure
quasiharmonic equations of state for dynamically-stabilized soft-mode materials
We introduce a method for treating soft modes within the analytical framework
of the quasiharmonic equation of state. The corresponding double-well
energy-displacement relation is fitted to a functional form that is harmonic in
both the low- and high-energy limits. Using density-functional calculations and
statistical physics, we apply the quasiharmonic methodology to solid periclase.
We predict the existence of a B1--B2 phase transition at high pressures and
temperatures
Spatial Patterns Induced Purely by Dichotomous Disorder
We study conditions under which spatially extended systems with coupling a la
Swift-Hohenberg exhibit spatial patterns induced purely by the presence of
quenched dichotomous disorder. Complementing the theoretical results based on a
generalized mean-field approximation, we also present numerical simulations of
particular dynamical systems that exhibit the proposed phenomenology
Noise-Driven Mechanism for Pattern Formation
We extend the mechanism for noise-induced phase transitions proposed by
Ibanes et al. [Phys. Rev. Lett. 87, 020601-1 (2001)] to pattern formation
phenomena. In contrast with known mechanisms for pure noise-induced pattern
formation, this mechanism is not driven by a short-time instability amplified
by collective effects. The phenomenon is analyzed by means of a modulated mean
field approximation and numerical simulations
Effects of Pore Walls and Randomness on Phase Transitions in Porous Media
We study spin models within the mean field approximation to elucidate the
topology of the phase diagrams of systems modeling the liquid-vapor transition
and the separation of He--He mixtures in periodic porous media. These
topologies are found to be identical to those of the corresponding random field
and random anisotropy spin systems with a bimodal distribution of the
randomness. Our results suggest that the presence of walls (periodic or
otherwise) are a key factor determining the nature of the phase diagram in
porous media.Comment: REVTeX, 11 eps figures, to appear in Phys. Rev.
Crystallization of a supercooled liquid and of a glass - Ising model approach
Using Monte Carlo simulations we study crystallization in the
three-dimensional Ising model with four-spin interaction. We monitor the
morphology of crystals which grow after placing crystallization seeds in a
supercooled liquid. Defects in such crystals constitute an intricate and very
stable network which separate various domains by tensionless domain walls. We
also show that the crystallization which occurs during the continuous heating
of the glassy phase takes place at a heating-rate dependent temperature.Comment: 7 page
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