•Consistency between two MPS approaches and between MPS and qPCR.•Greater detection of Bacillus at the Canberra Airport with MPS compared to qPCR.•Selected MPS target(s) generated metagenomic ...fingerprint of bacterial community.•Selected MPS target(s) could not resolve Bacillus beyond the B. cereus group.•Choice of target(s) is key to differentiate pathogenic from non-pathogenic species.
Anthrax, caused by the Gram-positive, spore forming bacterium Bacillus anthracis, is a disease with naturally occurring outbreaks in many parts of the world, primarily in domestic and wild herbivores. Due to the movement of people and stock, B. anthracis could, however, be at transportation hubs including airports. The continuous threat to national and international security from a biological agent release, or hoax attack, is a very real concern. Sensitive, robust and rapid (hours-day) methods to identify biological agents, including B. anthracis, and distinguish pathogenic from non-pathogenic species, is an essential cornerstone to national security. The aim of this project was to determine the presence of Bacillus species at the Canberra Airport using two massively parallel sequencing (MPS) approaches and compare with previous results using real-time polymerase chain reaction (qPCR).
Samples were collected daily for seven days each month from August 2011–July 2012 targeting movement of people, luggage and freight into and out of the Canberra Airport. Extracted DNA was analysed using qPCR specific for B. anthracis. A subset of samples was analysed using two MPS approaches. Approach one, using the Ion PGM™ (Thermo Fisher Scientific; TFS) and an in-house assay, targeted the two B. anthracis virulence plasmids (cya and capB genes) and a single conserved region of the 16S rRNA gene. Approach two, using the Ion S5™ (TFS) and the commercial Ion 16S™ Metagenomics Kit (TFS), targeted multiple regions within the bacterial 16S rRNA gene.
Overall there was consistency between the two MPS approaches and between MPS and qPCR, however, MPS was more sensitive, particularly for plasmid detection. Whilst the broad-range 16S genomic target(s) used in both MPS approaches in this study was able to generate a metagenomic fingerprint of the bacterial community at the Canberra Airport, it could not resolve Bacillus species beyond the level of the Bacillus cereus group. The inclusion of B. anthracis virulence plasmid targets in the in-house assay did allow for the potential presumptive identifications of pathogenic species. No plasmid targets were in the Ion 16S™ Metagenomics Kit.
This study shows the choice of target(s) is key in MPS assay development and should be carefully considered to ensure the assay is fit for purpose, whether as an initial screening (presumptive) or a more specific (but not entirely confirmatory) test. Identification approaches may also benefit from a combination of MPS and qPCR as each has benefits and limitations.
•New biological sources have been found to yield sufficient DNA and should be considered for DVI.•New sampling procedures can offer rapid and practical solutions to further expedite the DVI ...process.•New preservative solutions can preserve post mortem tissue, leech DNA into solution and are compatible with direct-to-PCR.•Emerging technologies could be applied to DVI to aid identification and provide investigative leads.•Guidelines should be revised to reflect current best practice for sample selection, collection, preservation and profiling.
DNA profiling has emerged as the gold standard for the identification of victims in mass disaster events providing an ability to identify victims, reassociate remains and provide investigative leads at a relatively low cost, and with a high degree of discrimination. For the majority of samples, DNA-based identification can be achieved in a fast, streamlined and high-throughput manner. However, a large number of remains will be extremely compromised, characteristic of mass disasters. Advances in technology and in the field of forensic biology have increased the options for the collection, sampling, preservation and processing of samples for DNA profiling. Furthermore, recent developments now allow a vast array of new genetic markers and genotyping techniques to extract as much genetic information from a sample as possible, ensuring that identification is not only accurate but also possible where material is degraded, or limited. Where historically DNA profiling has involved comparison with ante mortem samples or relatives, now DNA profiling can direct investigators towards putative victims or relatives, for comparison through the determination of externally visible characteristics, or biogeographical ancestry. This paper reviews the current and emerging tools available for maximising the recovery of genetic information from post mortem samples in a disaster victim identification context.
The Australian threat environment has changed as a result of global terrorism and foreign fighters, which are contributing factors to the blurring of the lines between military objectives and law ...enforcement activities. This shift requires a more integrated and interoperable forensic science framework that is coordinated at the whole-of-Government enterprise level. The forensic science 'system of systems' provides an integrating framework, which recognizes that forensic science supports the criminal justice, law enforcement, intelligence, and military systems. These systems must work together to achieve end-to-end performance, which is coordinated through integration and sharing of information across the systems. The purpose of this paper is to discuss why we need a systems thinking approach to military forensic science in the contemporary world. Implementation of a coordinated and interoperable military forensic science system of systems will contribute to the Australian Department of Defence strategic objective to deter, deny and defeat returned foreign fighters and global terrorist networks, such as Islamic State of Iraq and Syria (ISIS).
Over the last decade, reviews of the forensic science industry have addressed critical system failures and made recommendations for improvements to the industry. These reviews identify systemic risks ...and issues but they do not explicitly address the need for strategic risk and issue management systems to deal with the identified issues and to mitigate risks. Risk and issue management, along with quality management systems, are critical components of the forensic science 'system of systems', which describes forensic science as a supporting system in the larger criminal justice, law enforcement, intelligence, and military systems. The update of ISO 17,025:2017 General requirements for the competence of testing and calibration laboratories now includes a chapter on risk-based thinking. Furthermore, ISO 31,000 principle and guidelines for risk management was updated in 2018 and it is important for the forensic science industry to consider the updated ISO 17,025:2017 and ISO 31,000. In this paper, we discuss risk and issues management in relation to the forensic science system of systems. The purpose is to review the strategic risks identified in reviews of the forensic science industry to identify risk treatments to help to mitigate against critical system failure, such as miscarriages of justice.
The Improvised Explosive Device (IED) threat faced by coalition partners in Iraq and Afghanistan required concepts of operations to identify threat actors, network analysis to attack the network, and ...counter-IED strategies to defeat the devices. Forensic science was employed beyond its traditional law enforcement investigation and criminal justice objectives, and provided a capability for military organizations to help to counter IEDs. Forensic science in support of the expeditionary force on the battlefield is referred to as expeditionary forensic science, which is not well understood outside of the military domain. Military forensic science could be considered the black sheep of the forensic family as it is often left out or not considered alongside traditional forensic science. The purpose of this paper and plenary address, presented at the Australian and New Zealand Forensic Science Society (ANZFSS) 24th International Symposium, is to provide the broader forensic science community with a better understanding of military expeditionary forensic science. The aim is to promote greater partnerships between law enforcement, intelligence, the criminal justice system, and military forensic science as part of the broader forensic science 'system of systems'.
Abbreviation: System of systems (SOS); improvised explosive devices (IED)
Law enforcement forensic intelligence is the accurate, timely and useful product of logically processing forensic case data for investigation and/or intelligence purposes and is, to some extent, an ...emerging discipline. In the military domain, intelligence has a long history and military organizations have forensic science capabilities. However, to date, military organizations have not formally implemented forensic intelligence models. Forensic science is an end-to-end process and forensic intelligence spans the continuum between forensic science informing law enforcement investigations or military intelligence through to prosecution in the criminal justice system. Hence, forensic intelligence can be consistent with military objectives and could be a valuable model for military organizations in the long-term. The purpose of this article is to review law enforcement forensic intelligence models and explore requirements for military forensic intelligence models. To achieve operational utility, a military forensic intelligence model needs to enable the provision of timely forensic intelligence to be fused with all-source intelligence. The right methodologies will preserve the forensic science rigor, which will balance the forensic intelligence needs with the criminal justice system standards. A principle-based approach to the development of a military forensic intelligence model can provide a flexible and adaptive model across the spectrum of military conflicts.
Forensic science is undertaken in support of law enforcement investigations, criminal justice prosecutions, intelligence, and military objectives. There are different forensic operating models to ...meet these various objectives and no unified approach to forensic science has been described. There are common challenges in the military domain, which impacts the delivery of forensic science, including: different terminology between organizations, complex operating environments, operating siloes, inward-looking military structures, quality management, and resource constraints. One solution to these challenges is to apply modern organizational theory to military-focused forensic science. Organizational theory is the study of organizations in a structural sense, including objectives, people, structure and management. The modern organizational theory systems approach describes how organizations should be viewed as systems within larger systems. In the work presented in this paper, a systems approach has been applied, for the first time, to military forensic exploitation to address the common issues faced by military organizations. The advantages of applying a systems approach to military forensic exploitation are that it is applicable across forensic science organizations, it is flexible and scalable to meet the changing external environment, and it articulates the redundancies in the system to help address system failures.
Massively parallel sequencing (MPS) of identity informative single-nucleotide polymorphisms (IISNPs) enables hundreds of forensically relevant markers to be analysed simultaneously. Generating DNA ...sequence data enables more detailed analysis including identification of sequence variations between individuals. The GeneRead DNAseq 140 IISNP MPS panel (QIAGEN) has been evaluated on both the MiSeq (Illumina) and Ion PGM™ (Applied Biosystems) MPS platforms using the GeneRead DNAseq Targeted Panels V2 library preparation workflow (QIAGEN). The aims of this study were to (1) determine if the GeneRead DNAseq panel is effective for identity testing by assessing deviation from Hardy-Weinberg (HWE) and pairwise linkage equilibrium (LE); (2) sequence samples with the GeneRead DNAseq panel on the Ion PGM™ using the QIAGEN workflow and assess specificity, sensitivity and accuracy; (3) assess the efficacy of adding biological samples directly to the GeneRead DNAseq PCR, without prior DNA extraction; and (4) assess the effect of varying coverage and allele frequency thresholds on genotype concordance. Analyses of the 140 SNPs for HWE and LE using Fisher’s exact tests and the sequential Bonferroni correction revealed that one SNP was out of HWE in the Japanese population and five SNP combinations were commonly out of LE in 13 of 14 populations. The panel was sensitive down to 0.3125 ng of DNA input. A direct-to-PCR approach (without DNA extraction) produced highly concordant genotypes. The setting of appropriate allele frequency thresholds is more effective for reducing erroneous genotypes than coverage thresholds.
Abstract Interleukin-15 (IL-15) is a potent growth factor for activated T and natural killer (NK) cells, stimulator of memory T cells and plays an important role in viral immunity. To investigate ...mechanisms underlying the antiviral activity of IL-15, a recombinant vaccinia virus (rVV) encoding murine IL-15 (VV-IL-15) was constructed. Following infection of mice with VV-IL-15, virus titres in the ovaries were significantly reduced compared to mice infected with control VV. Growth of VV-IL-15 was also reduced in nude athymic mice, indicating the antiviral activity of IL-15 does not require T cells. Additionally, VV-IL-15 augmented the cytolytic activity of natural NK cells in the spleen and enhanced interferon (IFN) mRNA expression and transcription factors associated with IFN induction. Using knockout mice and antibody depletion studies, we showed for the first time that the control of VV-IL-15 replication in mice is dependent on NK cells and IFNs and, in their absence, the protective role of IL-15 is abolished.
To investigate the contribution of the alternative sigma factor Sigma B to detergent stress in Listeria monocytogenes upon exposure to the surface-active agents, benzalkoniumchloride (BC), ...cetylpyridinium chloride (CPC) and sodium docecyl sulfate (SDS). Surfactant/detergent stress in L. monocytogenes 10403S and a ΔsigB mutant of 10403S was investigated by lethality, impact on growth and by transcriptional analysis. We observed a significant 1-2 log decrease in the viability of the ΔsigB mutant in response to lethal levels of surfactants. Transcriptional (reverse transcriptase-PCR) analysis revealed the induction of sigmaB by sublethal levels of surface-active agents. However, Sigma B does not play a significant role in the growth of L. monocytogenes upon exposure to sublethal levels as investigated by growth analysis. Sigma B is essential for the resistance of L. monocytogenes at lethal levels of BC, CPC and SDS. We demonstrate that Sigma B is essential for the resistance of the pathogen to surfactant stress. The findings raise the possibility that induction of Sigma B by sublethal levels of industrial cleaning agents may promote enhanced resistance of further food-processing associated stresses or conditions encountered during infection. Sigma B-regulated mechanisms of detergent resistance may provide targets for the future design of novel cleaning agents.
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