Development of rapid and highly resolving combinatorial genotyping schemes for Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni followed by Campylobacter coli contribute substantially to the economic and public health burden attributed to food-borne infections in Australia. Genotypic characterisation of isolates has provided new insights into the epidemiology and pathogenesis of C. jejuni and C. coli. However, currently available methods are not conducive to large scale epidemiological investigations that are necessary to elucidate the global epidemiology of these common food-borne pathogens. This research aims to develop high resolution C. jejuni and C. coli genotyping schemes that are convenient for high throughput applications. Real-time PCR and High Resolution Melt (HRM) analysis are fundamental to the genotyping schemes developed in this study and enable rapid, cost effective, interrogation of a range of different polymorphic sites within the Campylobacter genome. While the sources and routes of transmission of campylobacters are unclear, handling and consumption of poultry meat is frequently associated with human campylobacteriosis in Australia. Therefore, chicken derived C. jejuni and C. coli isolates were used to develop and verify the methods described in this study. The first aim of this study describes the application of MLST-SNP (Multi Locus Sequence Typing Single Nucleotide Polymorphisms) + binary typing to 87 chicken C. jejuni isolates using real-time PCR analysis. These typing schemes were developed previously by our research group using isolates from campylobacteriosis patients. This present study showed that SNP + binary typing alone or in combination are effective at detecting epidemiological linkage between chicken derived Campylobacter isolates and enable data comparisons with other MLST based investigations. SNP + binary types obtained from chicken isolates in this study were compared with a previously SNP + binary and MLST typed set of human isolates. Common genotypes between the two collections of isolates were identified and ST-524 represented a clone that could be worth monitoring in the chicken meat industry. In contrast, ST-48, mainly associated with bovine hosts, was abundant in the human isolates. This genotype was, however, absent in the chicken isolates, indicating the role of non-poultry sources in causing human Campylobacter infections. This demonstrates the potential application of SNP + binary typing for epidemiological investigations and source tracing. While MLST SNPs and binary genes comprise the more stable backbone of the Campylobacter genome and are indicative of long term epidemiological linkage of the isolates, the development of a High Resolution Melt (HRM) based curve analysis method to interrogate the hypervariable Campylobacter flagellin encoding gene (flaA) is described in Aim 2 of this study. The flaA gene product appears to be an important pathogenicity determinant of campylobacters and is therefore a popular target for genotyping, especially for short term epidemiological studies such as outbreak investigations. HRM curve analysis based flaA interrogation is a single-step closed-tube method that provides portable data that can be easily shared and accessed. Critical to the development of flaA HRM was the use of flaA specific primers that did not amplify the flaB gene. HRM curve analysis flaA interrogation was successful at discriminating the 47 sequence variants identified within the 87 C. jejuni and 15 C. coli isolates and correlated to the epidemiological background of the isolates. In the combinatorial format, the resolving power of flaA was additive to that of SNP + binary typing and CRISPR (Clustered regularly spaced short Palindromic repeats) HRM and fits the PHRANA (Progressive hierarchical resolving assays using nucleic acids) approach for genotyping. The use of statistical methods to analyse the HRM data enhanced sophistication of the method. Therefore, flaA HRM is a rapid and cost effective alternative to gel- or sequence-based flaA typing schemes. Aim 3 of this study describes the development of a novel bioinformatics driven method to interrogate Campylobacter MLST gene fragments using HRM, and is called ‘SNP Nucleated Minim MLST’ or ‘Minim typing’. The method involves HRM interrogation of MLST fragments that encompass highly informative “Nucleating SNPS” to ensure high resolution. Selection of fragments potentially suited to HRM analysis was conducted in silico using i) “Minimum SNPs” and ii) the new ’HRMtype’ software packages. Species specific sets of six “Nucleating SNPs” and six HRM fragments were identified for both C. jejuni and C. coli to ensure high typeability and resolution relevant to the MLST database. ‘Minim typing’ was tested empirically by typing 15 C. jejuni and five C. coli isolates. The association of clonal complexes (CC) to each isolate by ‘Minim typing’ and SNP + binary typing were used to compare the two MLST interrogation schemes. The CCs linked with each C. jejuni isolate were consistent for both methods. Thus, ‘Minim typing’ is an efficient and cost effective method to interrogate MLST genes. However, it is not expected to be independent, or meet the resolution of, sequence based MLST gene interrogation. ‘Minim typing’ in combination with flaA HRM is envisaged to comprise a highly resolving combinatorial typing scheme developed around the HRM platform and is amenable to automation and multiplexing. The genotyping techniques described in this thesis involve the combinatorial interrogation of differentially evolving genetic markers on the unified real-time PCR and HRM platform. They provide high resolution and are simple, cost effective and ideally suited to rapid and high throughput genotyping for these common food-borne pathogens.

The quest for authenticity in the west – negotiating the self in late modern cultures

Across western culture in the late modern era a number of phenomena have emerged that seek to challenge mainstream consumer capitalism and its effects on everyday lifestyles. Two of these movements labelled as Seachange (Amenity Migration) and Downshifting have grown steadily in popularity within the public sphere and also academic discourse. In this thesis these phenomena are investigated further using a Durkheimian platform for theoretical interrogation. It is argued that while previous research accomplishes much in the investigation of Seachange and Downshifting, there is a significant gap in theoretical explanation and synthesis that requires filling. Thus in this research, it is argued that the concept of self-authenticity assists in the fulfilment of this aim. It is shown here that authenticity guides the construction, negotiation and experience of the phenomena which serves to authenticate the self. It is further argued however that Downshifting and Seachange reflect a wider theme of the self where the individual seeks protection from the profane impacts of advanced capitalism. Subsequently, the thesis aims not only to reveal the underlying principles which feed each phenomenon, but also relate them back to a wider cultural narrative of the sacred self.

Towards trusted wireless sensor networks

This article presents the design and implementation of a trusted sensor node that provides Internet-grade security at low system cost. We describe trustedFleck, which uses a commodity Trusted Platform Module (TPM) chip to extend the capabilities of a standard wireless sensor node to provide security services such as message integrity, confidentiality, authenticity, and system integrity based on RSA public-key and XTEA-based symmetric-key cryptography. In addition trustedFleck provides secure storage of private keys and provides platform configuration registers (PCRs) to store system configurations and detect code tampering. We analyze system performance using metrics that are important for WSN applications such as computation time, memory size, energy consumption and cost. Our results show that trustedFleck significantly outperforms previous approaches (e.g., TinyECC) in terms of these metrics while providing stronger security levels. Finally, we describe a number of examples, built on trustedFleck, of symmetric key management, secure RPC, secure software update, and remote attestation.

Generic decoupled image-based visual servoing for cameras obeying the unified projection model

In this paper a generic decoupled imaged-based control scheme for calibrated cameras obeying the unified projection model is proposed. The proposed decoupled scheme is based on the surface of object projections onto the unit sphere. Such features are invariant to rotational motions. This allows the control of translational motion independently from the rotational motion. Finally, the proposed results are validated with experiments using a classical perspective camera as well as a fisheye camera mounted on a 6 dofs robot platform.

Virtual force feedback teleoperation of the InsectBot using optical flow

This paper proposes the use of optical flow from a moving robot to provide force feedback to an operator’s joystick to facilitate collision free teleoperation. Optical flow is measured by a pair of wide angle cameras on board the vehicle and used to generate a virtual environmental force that is reflected to the user through the joystick, as well as feeding back into the control of the vehicle. We show that the proposed control is dissipative and prevents the vehicle colliding with the environment as well as providing the operator with a natural feel for the remote environment. Experimental results are provided on the InsectBot holonomic vehicle platform.

Modelling and control of a quad-rotor robot

To date, most quad-rotor aerial robots have been based on flying toys. Although such systems can be used as prototypes, they are not sufficiently robust to serve as experimental robotics platforms. We have developed the X-4 Flyer, a quad-rotor robot using custom-built chassis and avionics with off-the-shelf motors and batteries, to be a highly reliable experimental platform. The vehicle uses tuned plant dynamics with an onboard embedded attitude controller to stabilise flight. A linear SISO controller was designed to regulate flyer attitude.

Poster abstract : Energy-efficient rate adaptive MAC Protocol (RA-MAC) for long-lived sensor networks

This paper introduces an energy-efficient Rate Adaptive MAC (RA-MAC) protocol for long-lived Wireless Sensor Networks (WSN). Previous research shows that the dynamic and lossy nature of wireless communication is one of the major challenges to reliable data delivery in a WSN. RA-MAC achieves high link reliability in such situations by dynamically trading off radio bit rate for signal processing gain. This extra gain reduces the packet loss rate which results in lower energy expenditure by reducing the number of retransmissions. RA-MAC selects the optimal data rate based on channel conditions with the aim of minimizing energy consumption. We have implemented RA-MAC in TinyOS on an off-the-shelf sensor platform (TinyNode), and evaluated its performance by comparing RA-MAC with state-ofthe- art WSN MAC protocol (SCP-MAC) by experiments.

Transforming agriculture through pervasive wireless sensor networks

A large-scale, outdoor, pervasive computing system based on the Fleck hardware platform applies sensor network technology to farming. Comprising static and animal-borne mobile nodes, the system measures the state of a complex, dynamic system comprising climate, soil, pasture, and animals. This data supports prediction of the land's future state and improved management outcomes through closed-loop control. This article is part of a special issue, Building a Sensor-Rich World.

Animal behaviour understanding using wireless sensor networks

This paper presents research that is being conducted by the Commonwealth Scientific and Industrial Research Organisation (CSIRO) with the aim of investigating the use of wireless sensor networks for automated livestock monitoring and control. It is difficult to achieve practical and reliable cattle monitoring with current conventional technologies due to challenges such as large grazing areas of cattle, long time periods of data sampling, and constantly varying physical environments. Wireless sensor networks bring a new level of possibilities into this area with the potential for greatly increased spatial and temporal resolution of measurement data. CSIRO has created a wireless sensor platform for animal behaviour monitoring where we are able to observe and collect information of animals without significantly interfering with them. Based on such monitoring information, we can identify each animal's behaviour and activities successfully

Data collection, storage and retrieval with an underwater sensor network

In this paper we present a novel platform for underwater sensor networks to be used for long-term monitoring of coral reefs and �sheries. The sensor network consists of static and mobile underwater sensor nodes. The nodes communicate point-to-point using a novel high-speed optical communication system integrated into the TinyOS stack, and they broadcast using an acoustic protocol integrated in the TinyOS stack. The nodes have a variety of sensing capabilities, including cameras, water temperature, and pressure. The mobile nodes can locate and hover above the static nodes for data muling, and they can perform network maintenance functions such as deployment, relocation, and recovery. In this paper we describe the hardware and software architecture of this underwater sensor network. We then describe the optical and acoustic networking protocols and present experimental networking and data collected in a pool, in rivers, and in the ocean. Finally, we describe our experiments with mobility for data muling in this network.

Towards dynamically-favourable quad-rotor aerial robots

This paper outlines progress towards realising practical quad-rotor robot helicopters and, in particular, the Australian National University’s ‘X-4 Flyer’ platform. Two challenges facing the X-4 are generating sufficient thrust and managing unstable dynamic behaviour. We address these issues with a rotor design technique for maximising thrust and the application of a novel rotor mast configuration. An aero-elastic blade design is described and its performance results are presented. A sprung teetering rotor hub that allows adjustment of the blade flapping characteristics and a quad-rotor dynamic model with blade flapping are introduced. The use of inverted rotors is shown to produce favorable stability properties for the Mark II X-4 Flyer.

DDX : A distributed software architecture for robotic systems

The Dynamic Data eXchange (DDX) is our third generation platform for building distributed robot controllers. DDX allows a coalition of programs to share data at run-time through an efficient shared memory mechanism managed by a store. Further, stores on multiple machines can be linked by means of a global catalog and data is moved between the stores on an as needed basis by multi-casting. Heterogeneous computer systems are handled. We describe the architecture of DDX and the standard clients we have developed that let us rapidly build complex control systems with minimal coding.

Vibration isolation for autonomous helicopter flight

The mechanisms of helicopter flight create a unique, high-vibration environment which can play havoc with the accurate operation of on-board sensors. Vibration isolation of electronic sensors from structural borne oscillations is paramount to their reliable and accurate use. Effective isolation is achieved by realising a trade-off between the properties of the suspended instrument package, and the isolation mechanism. This is made more difficult as the weight and size of the sensors and computing hardware decreases with advances in technology. This paper presents a history of the design, challenges, constraints and construction of an integrated isolated vision and sensor platform and landing gear for the CSIRO autonomous X-Cell helicopter. The results of isolation performance and in-flight tests of the platform in autonomous flight are presented.

Visual servoing of a car-like vehicle - an application of omnidirectional vision

In this paper, we develop the switching controller presented by Lee et al. for the pose control of a car-like vehicle, to allow the use of an omnidirectional vision sensor. To this end we incorporate an extension to a hypothesis on the navigation behaviour of the desert ant, cataglyphis bicolor, which leads to a correspondence free landmark based vision technique. The method we present allows positioning to a learnt location based on feature bearing angle and range discrepancies between the robot's current view of the environment, and that at a learnt location. We present simulations and experimental results, the latter obtained using our outdoor mobile platform.

Experiments in learning helicopter control from a pilot

This paper details the development of a machine learning system which uses the helicopter state and the actions of an instructing pilot to synthesise helicopter control modules online. Aggressive destabilisation/restabilisation sequences are used for training, such that a wide state space envelope is covered during training. The performance of heading, roll, pitch, height and lateral velocity control learning is presented using our Xcell 60 experimental platform. The helicopter is demonstrated to be stabilised on all axes using the “learning from a pilot” technique. To our knowledge, this is the first time a “learning from a pilot” technique has been successfully applied to all axes.