Application of high-throughput sequencing to whole rabies viral genome characterisation and its use for phylogenetic re-evaluation of a raccoon strain incursion into the province of Ontario

Raccoon rabies remains a serious public health problem throughout much of the eastern seaboard of North America due to the urban nature of the reservoir host and the many challenges inherent in multi-jurisdictional efforts to administer co-ordinated and comprehensive wildlife rabies control programmes. Better understanding of the mechanisms of spread of rabies virus can play a significant role in guiding such control efforts. To facilitate a detailed molecular epidemiological study of raccoon rabies virus movements across eastern North America, we developed a methodology to efficiently determine whole genome sequences of hundreds of viral samples. The workflow combines the generation of a limited number of overlapping amplicons covering the complete viral genome and use of high throughput sequencing technology. The value of this approach is demonstrated through a retrospective phylogenetic analysis of an outbreak of raccoon rabies which occurred in the province of Ontario between 1999 and 2005. As demonstrated by the number of single nucleotide polymorphisms detected, whole genome sequence data were far more effective than single gene sequences in discriminating between samples and this facilitated the generation of more robust and informative phylogenies that yielded insights into the spatio-temporal pattern of viral spread. With minor modification this approach could be applied to other rabies virus variants thereby facilitating greatly improved phylogenetic inference and thus better understanding of the spread of this serious zoonotic disease. Such information will inform the most appropriate strategies for rabies control in wildlife reservoirs

Processes underlying rabies virus incursions across US–Canada Border as revealed by whole-genome phylogeography

Disease control programs aim to constrain and reduce the spread of infection. Human disease interventions such as wildlife vaccination play a major role in determining the limits of a pathogen’s spatial distribution. Over the past few decades, a raccoon-specific variant of rabies virus (RRV) has invaded large areas of eastern North America. Although expansion into Canada has been largely prevented through vaccination along the US border, several outbreaks have occurred in Canada. Applying phylogeographic approaches to 289 RRV whole-genome sequences derived from isolates collected in Canada and adjacent US states, we examined the processes underlying these outbreaks. RRV incursions were attributable predominantly to systematic virus leakage of local strains across areas along the border where vaccination has been conducted but also to single stochastic events such as long-distance translocations. These results demonstrate the utility of phylogeographic analysis of pathogen genomes for understanding transboundary outbreaks

Impacts of removing badgers on localised counts of hedgehogs

This is the final version of the article. Available from Public Library of Science via the DOI in this record.Experimental evidence of the interactions among mammalian predators that eat or compete with one another is rare, due to the ethical and logistical challenges of managing wild populations in a controlled and replicated way. Here, we report on the opportunistic use of a replicated and controlled culling experiment (the Randomised Badger Culling Trial) to investigate the relationship between two sympatric predators: European badgers Meles meles and western European hedgehogs Erinaceus europaeus. In areas of preferred habitat (amenity grassland), counts of hedgehogs more than doubled over a 5-year period from the start of badger culling (from 0.9 ha-1 pre-cull to 2.4 ha-1 post-cull), whereas hedgehog counts did not change where there was no badger culling (0.3-0.3 hedgehogs ha-1). This trial provides experimental evidence for mesopredator release as an outcome of management of a top predator.The study was funded by the United Kingdom Government’s Department for Environment, Food and Rural Affairs (http://www.defra.gov.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Three studies showing the importance of quantitative methods in investigation of veterinary infectious disease

Chapter 1 – EXAMINING THE EVOLUTIONARY HISTORY OF BOVINE PAPILLOMAVIRUS IN EQUINE SARCOIDS The papillomavirus (PV) family consist of slowly evolving host-adapted DNA viruses. Bovine papillomaviruses (BPVs) -1 and -2 primarily cause warts in their natural host, the cow, but also lead to locally aggressive and invasive skin tumours in equids known as sarcoids. This chapter gives an account of the first phylogenetic analysis of BPV in equine sarcoids, undertaken in order to clarify the evolutionary history of the virus and its cross-species association with equine sarcoids. Phylogenetic trees were constructed for three different stretches of the BPV genome. Although two of these analyses used gene segments that proved too short to draw any firm conclusions, the phylogenetic analysis carried out on the BPV-1 transcriptional promoter region (LCR) from cattle and horse samples provided interesting insights into the evolution of the virus. The genetic diversity seen in the LCR variants was shown to be ancient, predating domestication of both equids and cattle. The phylogenetic tree shows clear geographic segregation, with an ancestral BPV-1 group consisting of African and Brazilian sequences and a more evolved European group of sequences. The distribution of the cattle samples within the phylogeny suggests the sequences originally evolved in ancestral cattle, and that the genetic diversity found in equine sarcoids is the result of multiple, relatively recent species jumps into horses from different seeding strains of the virus. In addition, a specific LCR sequence variant was isolated in equine samples from all countries sampled here, despite being absent from cattle samples, suggesting that viruses containing this sequence variant may have a selective advantage within the equine population. Chapter 2 - SCOTTISH SHEEP MOVEMENTS AND THEIR POTENTIAL FOR DISEASE TRANSMISSION Animal movements play a major role in the spread of livestock diseases. By identifying farms pivotal to the network of livestock movements, it may be possible to more efficiently curb the spread of disease. Diseases transmit over great ranges of timescale and infectiousness. Sheep are moved from premises to premises for a variety of different reasons and with widely varying residence times on the arrival premises, and different types of movement are important in the spread of different diseases. This report describes work identifying those sheep farms important in terms of the types of movements involved in both a fast-transmitting and a slowly-transmitting disease. In so doing it raises the possibility of achieving control of multiple infections by targeting just a single subset of farms. If this were possible it would provide a cost effective and efficient method to reduce the burden of disease in the national flock. Chapter 3 – THE IMPLICATIONS OF POST-INFECTION IMMUNITY FOR THE EPIDEMIOLOGY AND CONTROL OF Escherichia coli O157 INFECTION OF CATTLE This report describes the use of epidemiological modelling to investigate how a period of post-infection immunity impacts the transmission dynamics of E. coli O157. Shigatoxigenic strains of E. coli, including the O157 strain, cause severe disease in man despite being asymptomatic in cattle, their natural reservoir host. Previous work modelling the transmission dynamics of E. coli has assumed that an animal becomes immediately susceptible on recovery from an infection, but recent experimental evidence indicates this may not be the case. In this project, stochastic models were developed for E. coli in cattle, allowing comparison of the effects of a period of post-infection immunity with the previously used assumption of immediate return to susceptibility. The results show that post-infection immunity gives lower values for outbreak duration, and for mean and variance in prevalence, and that this is observed over a biologically plausible range of the basic reproduction number, Ro. This in turn indicates that E. coli infection is likely to be more difficult to control if post-infection immunity exists, especially at higher infection prevalences. This study also reveals that if the assumption of post-infection immunity is valid, an even higher degree of individual heterogeneity in transmission is needed to explain the degree of variance in E. coli O157 prevalence seen in the field, thus validating previous work which demonstrated the importance of supershedder animals and individual heterogeneity. This study provides the first steps in investigating how a period of immunity following E. coli infection of cattle affects conclusions drawn by previous work assuming an immediate return to susceptibility. Models allowing the incorporation of individual heterogeneity are needed to further investigate the subject

Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level

Background: Mycobacterium avium subsp. paratuberculosis (MAP), the causative bacterium of Johne’s disease in dairy cattle, is widespread in the Canadian dairy industry and has significant economic and animal welfare implications. An understanding of the population dynamics of MAP can be used to identify introduction events, improve control efforts and target transmission pathways, although this requires an adequate understanding of MAP diversity and distribution between herds and across the country. Whole genome sequencing (WGS) offers a detailed assessment of the SNP-level diversity and genetic relationship of isolates, whereas several molecular typing techniques used to investigate the molecular epidemiology of MAP, such as variable number of tandem repeat (VNTR) typing, target relatively unstable repetitive elements in the genome that may be too unpredictable to draw accurate conclusions. The objective of this study was to evaluate the diversity of bovine MAP isolates in Canadian dairy herds using WGS and then determine if VNTR typing can distinguish truly related and unrelated isolates.<p></p> Results: Phylogenetic analysis based on 3,039 SNPs identified through WGS of 124 MAP isolates identified eight genetically distinct subtypes in dairy herds from seven Canadian provinces, with the dominant type including over 80% of MAP isolates. VNTR typing of 527 MAP isolates identified 12 types, including “bison type” isolates, from seven different herds. At a national level, MAP isolates differed from each other by 1–2 to 239–240 SNPs, regardless of whether they belonged to the same or different VNTR types. A herd-level analysis of MAP isolates demonstrated that VNTR typing may both over-estimate and under-estimate the relatedness of MAP isolates found within a single herd.<p></p> Conclusions: The presence of multiple MAP subtypes in Canada suggests multiple introductions into the country including what has now become one dominant type, an important finding for Johne’s disease control. VNTR typing often failed to identify closely and distantly related isolates, limiting the applicability of using this typing scheme to study the molecular epidemiology of MAP at a national and herd-level.<p></p&gt

The Structure and Dynamics of Ions at Aqueous Interfaces Studied via Atomic Force Microscopy

The organisation and kinetics of charges at solid and soft interfaces play a central role in biological signalling processes and are vital for energy storage technologies as well as our understanding of heterogeneous catalysis. At the molecular-scale, such interfacial behaviour remains stubbornly difficult to characterise, due to the short-ranged interactions between ions, their aqueous solvent and surface groups. Thus, continuum-scale models quickly break down, especially close to the interface and with high charge densities. This thesis addresses the question of ionic organisation using atomic force microscopy (AFM), which uniquely combines sub-nanometre spatial resolution and the ability to probe relatively long timescales. The use of small oscillation amplitudes allows the topography of the ionic layer to be mapped while simultaneously extracting physical properties from the sample or the interface itself, with time resolution spanning from tens of milliseconds to minutes. The structure of ions at hydrophilic interfaces is shown to be delicately sensitive to the charges’ molecular structure (in the case of larger buffering agents) and their charge density (for simple alkali cations). Specifically, the cations’ interactions with a model lipid membrane and the waters around it lead to an attractive correlation energy which generates nanoscale networks that evolve over the course of many seconds. These ionic structures directly reduce the effective stiffness of the lipids, providing a mechanism for the spontaneous control of membranes’ mechanical properties. These ionic networks are significant in the case of confined fluids and provide an efficient means of lubrication even under high pressures in sub-nanometre gaps. When sheared, such fluid films are revealed to be non-Newtonian, with dynamics that depend on the velocity and lengthscale of the motion. The results highlight the greatly damped kinetics of ions and water molecules at interfaces, and shed light on the mechanisms behind their transport through and along biomolecules

Nanoscale probing of local dielectric changes at the interface between solids and aqueous saline solutions

The mobility of dissolved ions and charged molecules at interfaces underpins countless processes in science and technology. Experimentally, this is typically measured from the averaged response of the charges to an electrical potential. High-resolution Atomic Force Microscopy (AFM) can image single adsorbed ions and molecules at solid–liquid interfaces, but probing the associated dynamics remains highly challenging. One possible strategy is to investigate the response of the species of interest to a highly localized AC electric field in an approach analogous to dielectric spectroscopy. The dielectric force experienced by the AFM tip apex is modulated by the dielectric properties of the sample probed, itself sensitive to the mobilities of solvated charges and dipoles. Previous work successfully used this approach to quantify the dielectric constant of thin samples, but with limited spatial resolution. Here we propose a strategy to simultaneously map the nanoscale topography and local dielectric variations across a range of interfaces by conducting high-resolution AFM imaging concomitantly with electrical AC measurements in a multifrequency approach. The strategy is tested over a 500 MHz bandwidth in pure liquids with different dielectric constants and in saline aqueous solutions. In liquids with higher dielectric constants, the system behaves as inductive–resistive–capacitive but the adjunction of ions removes the inductive resonances and precludes measurements at higher frequencies. High-resolution imaging is demonstrated over single graphene oxide (GrO) flakes with simultaneous but decoupled dielectric measurements. The dielectric constant is consistent and reproducible across liquids, except at higher salt concentrations where frequency-dependent effects occur. The results suggest the strategy is suitable for nanometre-level mapping of the dielectric properties of solid–liquid interfaces, but more work is needed to fully understand the different physical effects underpinning the measurements

Local mapping of the nanoscale viscoelastic properties of fluid membranes by AFM nanorheology

Biological membranes are intrinsically dynamic entities that continually adapt their biophysical properties and molecular organisation to support cellular function. Current microscopy techniques can derive high-resolution structural information of labelled molecules but quantifying the associated viscoelastic behaviour with nanometre precision remains challenging. Here, we develop an approach based on atomic force microscopy in conjunction with fast nano-actuators to map the viscoelastic response of unlabelled supported membranes with nanometre spatial resolution. On fluid membranes, we show that the method can quantify local variations in the molecular mobility of the lipids and derive a diffusion coefficient. We confirm our experimental approach with molecular dynamics simulations, also highlighting the role played by the water at the interface with the membrane on the measurement. Probing ternary model bilayers reveals spatial correlations in the local diffusion over distances of ≈20 nm within liquid disordered domains. This lateral correlation is enhanced in native bovine lens membranes, where the inclusion of protein-rich domains induces four-fold variations in the diffusion coefficient across < 100 nm of the fluid regions, consistent with biological function. Our findings suggest that diffusion is highly localised in fluid biomembranes

The genetic and spatial epidemiology of bovine tuberculosis in the UK: from molecular typing to bacterial whole genome sequencing

Bovine tuberculosis (bTB) is a disease of cattle and other animals caused by the bacterium Mycobacterium bovis. In the UK, control of the disease presents significant difficulties, with bTB currently one of the most important diseases affecting the livestock industry in this region. The involvement of infection in a wildlife reservoir, the Eurasian badger Meles meles, is a key challenge to the eradication of bTB in the UK and causes much public controversy. In such a situation it is essential to use all available tools to control the spread of the disease. For many years, molecular typing of the M. bovis population has been routinely employed to understand the epidemiology of bTB in Britain and Northern Ireland (NI), providing broad scale information on the M. bovis population. More recently, high-resolution bacterial whole genome sequencing (WGS) of M. bovis has become feasible, although its use had yet to be explored in depth for the epidemiology of bTB. In this thesis, I describe various approaches to the use of pathogen genetic and spatial information to explore the epidemiology of bTB in the UK. I start at the broad scale, analysing the molecular types of M. bovis across the whole of NI, and go on to evaluate the use of WGS for M. bovis in targeted subpopulations of cattle and badgers. Following a general overview (Chapter 1), I explore the processes underlying the pattern of relative abundances of M. bovis molecular types in NI, showing that simple neutral processes are not capable of generating the distribution observed, and using simulation models to demonstrate that historical increases in bTB prevalence and/or transmission heterogeneity may be responsible (Chapter 2). In Chapter 3, I examine the spatial structure of the NI M. bovis population, demonstrating significant spatial clustering of M. bovis molecular types and correlations between the types present in cattle and badgers. I go on to develop a landscape genetics analysis which provides preliminary indications that transmission in badgers might be responsible for the spatial structure observed in M. bovis infections in cattle. In Chapter 4, I evaluate the use of high density bacterial WGS in M. bovis isolates belonging to a single molecular type. I use these data to demonstrate some of the potential of WGS, while also highlighting limitations inherent in using these approaches in such a slowly evolving pathogen. In Chapter 5, I take methods developed in the preceding chapters and apply them to the study of bTB in a well studied badger population, characterising the genetic diversity of M. bovis present in these badgers and their links to local cattle infections. As a whole, this body of work shows that there is much to be gained in our understanding of the epidemiology of bTB from genetic and genomic data, both from examining the large historical datasets that already exist on molecular types of M. bovis in the UK, as well as from the application of high resolution bacterial WGS in this system

Journeys to engagement with the UK global justice movement: life stories of activist-educators

This thesis explores how individuals in the UK come to and sustain engagement with global justice issues (such as poverty, development and human rights). It responds to a scarcity of relevant research and a stated desire for greater understanding from those involved in development education and related areas. Relevant literature is used to develop: a working definition of the UK Global Justice Movement; a new conceptual framework for understanding forms of engagement; a ‘route map’ summarising knowledge about individuals’ journeys to engagement; and an understanding of current practice and debates in development education and related fields. Using narrative research techniques, the study then presents five individuals’ life stories with respect to engagement with global justice issues. The respondents come from a range of backgrounds and utilise a number of different forms of engagement, but all act in some way as educators/multipliers of engagement. Their stories are analysed using two different ‘lenses’: together, considering themes relevant to development education, and separately, investigating how concepts related to identity (Social Identity Theory, Identity Theory and Narrative Identity) can be used to understand individuals’ engagement. This analysis includes discussion of: the places in which learning happens; debates concerning learning, criticality and visits overseas; the extent to which respondents might be understood to be development educators themselves; roles they have played; the in- (and out-) groups mentioned; and the various sources of narrative available to each of them over the course of their journeys to and within engagement. Finally, the thesis suggests implications for researchers, policy makers and practitioners. This includes: future use of the concepts developed; further exploration of the potential learning value of ‘low cost’ forms engagement; supporting individuals to engage with different organisations and issues ‘across’ the movement; and, considering possibilities for work with families and faith groups