Showing papers by "Defence Science and Technology Laboratory published in 2004"

Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei

Abstract: Burkholderia pseudomallei is a recognized biothreat agent and the causative agent of melioidosis. This Gram-negative bacterium exists as a soil saprophyte in melioidosis-endemic areas of the world and accounts for 20% of community-acquired septicaemias in northeastern Thailand where half of those affected die. Here we report the complete genome of B. pseudomallei, which is composed of two chromosomes of 4.07 megabase pairs and 3.17 megabase pairs, showing significant functional partitioning of genes between them. The large chromosome encodes many of the core functions associated with central metabolism and cell growth, whereas the small chromosome carries more accessory functions associated with adaptation and survival in different niches. Genomic comparisons with closely and more distantly related bacteria revealed a greater level of gene order conservation and a greater number of orthologous genes on the large chromosome, suggesting that the two replicons have distinct evolutionary origins. A striking feature of the genome was the presence of 16 genomic islands (GIs) that together made up 6.1% of the genome. Further analysis revealed these islands to be variably present in a collection of invasive and soil isolates but entirely absent from the clonally related organism B. mallei. We propose that variable horizontal gene acquisition by B. pseudomallei is an important feature of recent genetic evolution and that this has resulted in a genetically diverse pathogenic species.

ATP-binding cassette transporters are targets for the development of antibacterial vaccines and therapies.

Abstract: ATP-binding cassette (ABC) transporters are widespread among living organisms and comprise one of the largest protein families. For example, components of ABC transporters are encoded by approximately 5% of the Escherichia coli and Bacillus subtilis genomes ([47][1], [80][2]). These transporters are

Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa type III secretion mutant in murine models of melioidosis.

Abstract: Melioidosis is a severe infectious disease of animals and humans caused by the Gram-negative intracellular pathogen Burkholderia pseudomallei. An Inv/Mxi-Spa-like type III protein secretion apparatus, encoded by the B. pseudomallei bsa locus, facilitates bacterial invasion of epithelial cells, escape from endocytic vesicles and intracellular survival. This study investigated the role of the Bsa type III secretion system in the pathogenesis of melioidosis in murine models. B. pseudomallei bipD mutants, lacking a component of the translocation apparatus, were found to be significantly attenuated following intraperitoneal or intranasal challenge of BALB/c mice. Furthermore, a bipD mutant was attenuated in C57BL/6 IL-12 p40−/− mice, which are highly susceptible to B. pseudomallei infection. Mutation of bipD impaired bacterial replication in the liver and spleen of BALB/c mice in the early stages of infection. B. pseudomallei mutants lacking either the type III secreted guanine nucleotide exchange factor BopE or the putative effectors BopA or BopB exhibited varying degrees of attenuation, with mutations in bopA and bopB causing a significant delay in median time to death. This indicates that bsa-encoded type III secreted proteins may act in concert to determine the outcome of B. pseudomallei infection in mice. Mice inoculated with the B. pseudomallei bipD mutant were partially protected against subsequent challenge with wild-type B. pseudomallei. However, immunization of mice with purified BipD protein was not protective.

Clostridium Perfringens Epsilon-Toxin Shows Structural Similarity to the Pore-Forming Toxin Aerolysin

Abstract: e-Toxin from Clostridium perfringens is a lethal toxin. Recent studies suggest that the toxin acts via an unusually potent pore-forming mechanism. Here we report the crystal structure of e-toxin, which reveals structural similarity to aerolysin from Aeromonas hydrophila. Pore-forming toxins can change conformation between soluble and transmembrane states. By comparing the two toxins, we have identified regions important for this transformation.

Absence of Yersinia pestis-specific DNA in human teeth from five European excavations of putative plague victims

Abstract: This study reports the results of a collaborative study undertaken by two independent research groups to (a) confirm recent PCR-based detection of Yersinia pestis DNA in human teeth from medieval plague victims in France, and (b) to extend these observations over five different European burial sites believed to contain plague victims dating from the late 13th to 17th centuries. Several different sets of primers were used, including those previously documented to yield positive results on ancient DNA extracts. No Y. pestis DNA could be amplified from DNA extracted from 108 teeth belonging to 61 individuals, despite the amplification of numerous other bacterial DNA sequences. Several methods of extracting dentine prior to the DNA extraction were also compared. PCR for bacterial 16S rDNA indicated the presence of multiple bacterial species in 23 out of 27 teeth DNA extracts where dentine was extracted using previously described methods. In comparison, positive results were obtained from only five out of 44 teeth DNA extracts for which a novel contamination-minimizing embedding technique was used. Therefore, high levels of environmental bacterial DNA are present in DNA extracts where previously described methods of tooth manipulation are used. To conclude, the absence of Y. pestis-specific DNA in an exhaustive search using specimens from multiple putative European plague burial sites does not allow us to confirm the identification of Y. pestis as the aetiological agent of the Black Death and subsequent plagues. In addition, the utility of the published tooth-based ancient DNA technique used to diagnose fatal bacteraemias in historical epidemics still awaits independent corroboration.

Satisfaction with performance appraisal systems

Abstract: This study examines the inter‐relationship between a person's role as appraisee and/or appraiser in a performance appraisal system (PAS) and level of satisfaction expressed with the system. It builds on earlier US studies which found that being an appraiser and, therefore, also an appraisee increased a person's satisfaction level compared with those who were appraisees only. Data were gathered from 382 respondents to examine three aspects of organisational justice which are known to influence satisfaction with PASs. The results indicate no intrinsic difference in satisfaction level linked to role in administering a PAS and that the source of satisfaction and/or dissatisfaction with the PAS was not equally attributable to all aspects of organisational justice.

Evaluation of lipopolysaccharide and capsular polysaccharide as subunit vaccines against experimental melioidosis.

Abstract: Burkholderia pseudomallei is the causative agent of melioidosis, which is a major cause of morbidity and mortality in endemic regions. Currently there is no human vaccine against melioidosis. In this study, LPS or capsular polysaccharide was used to immunize BALB/c mice. The different polysaccharide antigens induced antibody responses. Mice vaccinated with LPS developed predominantly IgM and IgG3 responses. Contrastingly, mice vaccinated with capsular polysaccharide developed a predominantly IgG2b response. After immunization, mice were challenged by the intra-peritoneal route and an increased mean time to death was observed compared with unvaccinated controls. Immunization with LPS provided an optimal protective response. Mice challenged by the aerosol route showed a small increase in the mean time to death compared with the unvaccinated controls. The passive transfer of antigen from immunized into naive mice provided protection against a subsequent challenge. This study is the first time antigens protective by active immunization have been identified and suggests that polysaccharides have potential as vaccine candidates against melioidosis.

Immunization with the C-domain of α-toxin prevents lethal infection, localizes tissue injury, and promotes host response to challenge with Clostridium perfringens

Abstract: Clostridium perfringens gas gangrene is characterized by rapid tissue destruction, impaired host response, and, often, death. Phospholipase C (alpha -toxin) is the virulence factor most responsible for these pathologies. The present study investigated the efficacy of active immunization with the C-terminal domain of alpha -toxin (Cpa247-370) in a murine model of gas gangrene. Primary end points of the study were survival, progression of infection, and tissue perfusion. Secondary end points, which were based on findings of histologic evaluation of tissues, included the extent of tissue destruction and microvascular thrombosis, as well as the magnitude of the tissue inflammatory response. Survival among C-domain-immunized animals was significantly greater than that among sham-immunized control animals. Furthermore, immunization with the C-domain localized the infection and prevented ischemia of the feet. Histopathologic findings demonstrated limited muscle necrosis, reduced microvascular thrombosis, and enhanced granulocytic influx in C-domain-immunized mice. We conclude that immunization with the C-domain of phospholipase C is a viable strategy for the prevention of morbidity and mortality associated with C. perfringens gas gangrene.

Genetic Relationships and Evolution of Genotypes of Yellow Fever Virus and Other Members of the Yellow Fever Virus Group within the Flavivirus Genus Based on the 3′ Noncoding Region

Abstract: Genetic relationships among flaviviruses within the yellow fever (YF) virus genetic group were investigated by comparing nucleotide sequences of the 3' noncoding region (3'NCR). Size heterogeneity was observed between members and even among strains of the same viral species. Size variation between YF strains was due to duplications and/or deletions of repeated nucleotide sequence elements (RYF). West African genotypes had three copies of the RYF (RYF1, RYF2, and RYF3); the Angola and the East and Central African genotypes had two copies (RYF1 and RYF3); and South American genotypes had only a single copy (RYF3). Nucleotide sequence analyses suggest a deletion within the 3'NCR of South American genotypes, including RYF1 and RYF2. Based on studies with the French neurotropic vaccine strain, passage of a YF virus strain in cell culture can result in deletion of RYF1 and RYF2. Taken together, these observations suggest that South American genotypes of YF virus evolved from West African genotypes and that the South American genotypes lost RYF1 and RYF2, possibly in a single event. Repeated sequence elements were found within the 3'NCR of other members of the YF virus genetic group, suggesting that it is probably characteristic for members of the YF virus genetic group. A core sequence of 15 nucleotides, containing two stem-loops, was found within the 3'NCR of all members of the YF genetic group and may represent the progenitor repeat sequence. Secondary structure predictions of the 3'NCR showed very similar structures for viruses that were closely related phylogenetically.

Analysis of β-Lyase Metabolites of Sulfur Mustard in Urine by Electrospray Liquid Chromatography-Tandem Mass Spectrometry

Abstract: A method is described for the analysis of b-lyase metabolites of sulfur mustard, 1-methylsulfinyl-2- [2-(methylthio)ethylsulfonyl]ethane and 1,1'-sulfonylbis [2-(methylsulfinyl)ethane], in human urine. The analytes were concentrated from urine on an ENV+ solid-phase extraction cartridge and analyzed by liquid chromatography-positive ion electrospray-tandem mass spectrometry in the selected reaction monitoring mode. Quantitation was performed against deuterated internal standards. Limits of detection were 0.1-0.5 ng/mL. The metabolites were detected in samples of urine from human casualties of sulfur mustard poisoning. The method provides a simpler alternative to gas chromatography-tandem mass spectrometry analysis, avoiding the need for reduction to less polar analytes.

Age influences resistance of Caenorhabditis elegans to killing by pathogenic bacteria.

Abstract: Caenorhabditis elegans has previously been proposed as an alternative host for models of infectious disease caused by human pathogens. When exposed to some human pathogenic bacteria, the life span of nematodes is significantly reduced. We have shown that mutations in the age-1, and/or age-2 genes of C. elegans, that normally enhance life expectancy, can also increase resistance to killing by the bacterial pathogens Pseudomonas aeruginosa, Salmonella enterica var. Typhimurium, Burkholderia cepacia or Yersinia pseudotuberculosis. We also found that the rate at which wild-type C. elegans was killed by the bacterial pathogens tested increased as nematodes aged. In the case of P. aeruginosa infection, the difference in life span of wild type and age-1 mutants of C. elegans was not due to differences in the level of bacterial colonisation of the gut.

A second survey of high explosives traces in public places.

Abstract: This survey was carried out as a follow-up to a 1994 survey carried out by this laboratory (1) in order to determine the background levels of explosives traces in public places. The first survey concentrated on transport areas and police stations in and around London. This second study examines levels in four of the United Kingdom's major cities: Birmingham, Cardiff, Glasgow, and Manchester. Samples were taken at various transport sites and from hotels, private houses, private vehicles, and clothing. The survey showed that traces of the high explosives nitroglycerine (NG), trinitrotoluene (TNT), pentaerythritol tetranitrate (PETN), and cyclotrimethylene trinitramine (RDX) are rare within the general public environment. Only one low-level trace of RDX was detected. NG, possibly associated with the use of firearms, was detected at low levels in two samples and 2,4-DNT was detected in a separate sample. No PETN was detected in any of the samples. The results of the survey indicate that it is unlikely that persons visiting public areas could become significantly contaminated with explosives. The analytical procedures employed would also have detected ethylene glycol dinitrate (EGDN) if present at levels greater than 2 ng, nitrobenzene (NB) if present at levels greater than 50 ng, mononitrotoluenes if present at levels greater than 50 ng, and the other common isomers of dinitrotoluene if these had been present at levels in excess of 10 ng. None of these were detected. The relatively high volatility of EGDN, NB, and the mononitrotoluenes would, however, cause traces of these compounds to disperse rapidly. A proportion of the samples (approximately 7%) were analyzed for the presence of HMX. No HMX was detected.

Joint Urban 2003 street canyon experiment

Abstract: As part of the DOE-DTRA Joint URBAN 2003 field experiment in Oklahoma City, a multi-group team instrumented a downtown street canyon with a high density of wind sensor instrumentation. The goal was to garner flow field information in order to better understand the transport and dispersion of tracers released in the street canyon. In this paper, we briefly review prior field and laboratory experiments on street canyon flow, describe our experimental set-up and measurement apparatus, present some preliminary analyses of the measurements, and discuss their significance in relation to current understanding. Eventually this data set will be used to evaluate the next generation of urban dispersion models (e.g., Cox et al., 2000; DeCroix, 2002; Hall et al., 2000; Williams et al., 2002).

Analysis of the sulfur mustard metabolite 1,1'-sulfonylbis[2-S-(N-acetylcysteinyl)ethane] in urine by negative ion electrospray liquid chromatography- tandem mass spectrometry.

Abstract: A method is described for the analysis of the sulfur mustard metabolite 1,1'-sulfonylbis[2-S-(N-acetylcysteinyl)ethane] in human urine. The analyte was concentrated from urine on a polymeric SPE cartridge and analyzed by liquid chromatography-negative ion electrospray tandem mass spectrometry in the selected reaction monitoring mode. The limit of detection was 0.5-1 ng/mL. The metabolite was detected at concentrations close to the detection limit in samples of urine from two casualties accidentally exposed to sulfur mustard from a First World War munition.

Protection Afforded by Heat Shock Protein 60 from Francisella tularensis Is Due to Copurified Lipopolysaccharide

Abstract: Heat shock proteins (Hsps) have attracted significant attention as protective antigens against a range of diseases caused by bacterial pathogens. However, more recently there have been suggestions that the protective response is due to the presence of peptide components other than Hsps. We have shown that mice that had been immunized with purified heat shock protein 60 (Hsp60) isolated from Francisella tularensis were protected against a subsequent challenge with some strains of the bacterium. However, this protection appeared to be due to trace amounts of lipopolysaccharide, which were too low to be detected by using the Limulus amoebocyte lysate assay. This finding raises the possibility that the protection afforded by other bacterial Hsp60 proteins may be due to trace quantities of polysaccharide antigens carried by and acting in conjunction with the Hsps.

Effect of temperature on skeletal muscle energy turnover during dynamic knee extensor exercise in humans

Abstract: The present study examined the effect of elevated temperature on muscle energy turnover during dynamic exercise. Nine male subjects performed 10 min of dynamic knee-extensor exercise at an intensit...

Second and third generation plague vaccines.

Abstract: A defined sub-unit vaccine against plague now seems to be feasible. The further development of this vaccine will be dependent on clinical trials with this vaccine. In parallel, there is good reason to believe that next-generation vaccines, will be given orally or intranasally.

A Study Into Commanders’ Understanding Of, And Attitudes To, Stress And Stress-Related Problems

Abstract: Objectives To undertake a tri-Service questionnaire survey to examine the commanders’ understanding of, and attitudes to, stress and stress-related problems. Methods A questionnaire was designed to elicit information on: Personal experience of stress and stress-related problems; Stress education; Pre-deployments briefings; and Post-incident stress debriefing. A total of 9,020 questionnaires were distributed between the three Services based on their proportional manning contribution to the Armed Forces as a whole. The population sampled ranged in rank from Corporal/Leading Rate up to, and including, Colonel/Captain RN/Group Captain (ranks below Leading Rate/Corporal were excluded because of their lack of command experience). The overall response rate was 55.8%. The study was carried out between September and December 2001 (i.e. pre-OP TELIC). Results Chronic work-based stressors were seen as most stressful when compared with family and health stressors. Most respondents accepted that stress and stress-related problems exist, but were reluctant to disclose their own stressrelated problems or seek help for fear that it might be detrimental both personally and professionally. There was found to be little support from peers or commanders. Little stress training was provided during recruit training, there were gaps in pre-deployment briefings and little in the way of post-deployment stress support. Conclusions Some of the more negative findings could have implications in terms of seeking help for stress-related problems at an early stage, which is counterproductive to the military’s genuine attempts to foster the psychological welfare of its employees. Some concerns could be alleviated by better and more timely stress education, preferably early on in a commander’s career, so that positive attitudes to stress and stressrelated problems can be formed and any negative attitudes changed, thereby bringing about a change in organisational culture in relation to stress. Some of the study’s concerns were addressed by the Operational Health Strategic Surveillance Committee which advised on operational health aspects of OP TELIC.

Validation of the mission-based approach to representing command and control in simulation models of conflict

Abstract: There is a need to represent military command and control in closed-form simulation models of conflict, in order to compare investment in such capability with alternative defence investments. This paper considers such representation of military command and control in the context of embodied cognitive science. This means that we represent such processes in terms of both decision-making and resultant behaviour. Previous work leads to the view that such a representation can be captured by a combination of deliberate (top down) planning and rapid (bottom up) planning. We have developed an approach on these lines as a way of representing human decision-making and behaviour in conflict. Here we show, by comparing simulation model results with real conflict situations, that our approach yields emergent force behaviour which is valid and representative. This thus increases our confidence that our representation of command and control in such simulation models is sufficient for our requirements.

Passive stabilization of a passively mode-locked laser by nonlinear absorption in indium phosphide

Abstract: A diode-pumped Nd:KGd(WO4)2 laser mode locked by a saturable Bragg reflector (SBR) is passively stabilized to suppress Q-switched mode locking and to extend the parameter range of continuous-wave mode locking An indium phosphide plate exhibiting two-photon absorption and free-carrier absorption is used for passive stabilization The intracavity pulse energy for the onset of stable continuous-wave mode locking is reduced by a factor of 4 compared with the laser without stabilization By increasing the modulation depth of the SBR, pulse shortening by 30% is achieved and bandwidth-limited 62-ps pulses are measured

Operational research in the UK Ministry of Defence: an overview

Abstract: The paper presents a survey of operational research (OR) as it is currently used in the UK Ministry of Defence (MoD). After a short historical introduction summarising developments since 1945, it identifies the impact which the end of the Cold War has had on defence operational analysis (OA), as the discipline is generally called within MoD, in terms of the problems studied, the technical challenges presented and the techniques employed. The factors, both external and internal, that have led to the MoD maintaining and indeed expanding its OA activity against a background of falling defence budgets are discussed in the context of previously reported research on the survival and success of OR groups.

The potential use of recombinant activated factor VII in trauma and surgery.

Abstract: It is possible that recombinant activated factor VII (rFVIIa) could revolutionise the medical and surgical management of haemorrhage following trauma and surgery due to its ease of administration and mechanism of action. This article reviews the evidence for the use of rFVIIa as a pro-coagulant, its mechanism of action, safety and recent research into its use in blunt and penetrating trauma and haemorrhage. The potential role of rFVIIa, both in the pre-hospital environment and in the emergency room or operating theatre, is discussed. Administration of rFVIIa at the roadside, on the battlefield or in a trauma centre may have significant potential implications, especially for the paramedical and surgical teams and even for strategic planners. Finally, the areas where further research is needed to provide objective evidence of its efficacy are elaborated. The potential thromboembolic complications of systemic administration of rFVIIa need to be carefully monitored but the high cost of this drug is likely to be the limiting factor in its widespread use.