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

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

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

A new phylodynamic model of Mycobacterium bovis transmission in a multi-host system uncovers the role of the unobserved reservoir

Multi-host pathogens are particularly difficult to control, especially when at least one of the hosts acts as a hidden reservoir. Deep sequencing of densely sampled pathogens has the potential to transform this understanding, but requires analytical approaches that jointly consider epidemiological and genetic data to best address this problem. While there has been considerable success in analyses of single species systems, the hidden reservoir problem is relatively under-studied. A well-known exemplar of this problem is bovine Tuberculosis, a disease found in British and Irish cattle caused by Mycobacterium bovis, where the Eurasian badger has long been believed to act as a reservoir but remains of poorly quantified importance except in very specific locations. As a result, the effort that should be directed at controlling disease in badgers is unclear. Here, we analyse densely collected epidemiological and genetic data from a cattle population but do not explicitly consider any data from badgers. We use a simulation modelling approach to show that, in our system, a model that exploits available cattle demographic and herd-to-herd movement data, but only considers the ability of a hidden reservoir to generate pathogen diversity, can be used to choose between different epidemiological scenarios. In our analysis, a model where the reservoir does not generate any diversity but contributes to new infections at a local farm scale are significantly preferred over models which generate diversity and/or spread disease at broader spatial scales. While we cannot directly attribute the role of the reservoir to badgers based on this analysis alone, the result supports the hypothesis that under current cattle control regimes, infected cattle alone cannot sustain M. bovis circulation. Given the observed close phylogenetic relationship for the bacteria taken from cattle and badgers sampled near to each other, the most parsimonious hypothesis is that the reservoir is the infected badger population. More broadly, our approach demonstrates that carefully constructed bespoke models can exploit the combination of genetic and epidemiological data to overcome issues of extreme data bias, and uncover important general characteristics of transmission in multi-host pathogen systems

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

Relative abundance of Mycobacterium bovis molecular types in cattle:a simulation study of potential epidemiological drivers

Background: The patterns of relative species abundance are commonly studied in ecology and epidemiology to provide insights into underlying dynamical processes. Molecular types (MVLA-types) of Mycobacterium bovis, the causal agent of bovine tuberculosis, are now routinely recorded in culture-confirmed bovine tuberculosis cases in Northern Ireland. In this study, we use ecological approaches and simulation modelling to investigate the distribution of relative abundances of MVLA-types and its potential drivers. We explore four biologically plausible hypotheses regarding the processes driving molecular type relative abundances: sampling and speciation; structuring of the pathogen population; historical changes in population size; and transmission heterogeneity (superspreading). Results: Northern Irish herd-level MVLA-type surveillance shows a right-skewed distribution of MVLA-types, with a small number of types present at very high frequencies and the majority of types very rare. We demonstrate that this skew is too extreme to be accounted for by simple neutral ecological processes. Simulation results indicate that the process of MVLA-type speciation and the manner in which the MVLA-typing loci were chosen in Northern Ireland cannot account for the observed skew. Similarly, we find that pathogen population structure, assuming for example a reservoir of infection in a separate host, would drive the relative abundance distribution in the opposite direction to that observed, generating more even abundances of molecular types. However, we find that historical increases in bovine tuberculosis prevalence and/or transmission heterogeneity (superspreading) are both capable of generating the skewed MVLA-type distribution, consistent with findings of previous work examining the distribution of molecular types in human tuberculosis. Conclusion: Although the distribution of MVLA-type abundances does not fit classical neutral predictions, our simulations show that increases in pathogen population size and/or superspreading are consistent with the pattern observed, even in the absence of selective pressures acting on the system

Whole Genome Sequencing Reveals Local Transmission Patterns of Mycobacterium bovis in Sympatric Cattle and Badger Populations

Whole genome sequencing (WGS) technology holds great promise as a tool for the forensic epidemiology of bacterial pathogens. It is likely to be particularly useful for studying the transmission dynamics of an observed epidemic involving a largely unsampled ‘reservoir' host, as for bovine tuberculosis (bTB) in British and Irish cattle and badgers. BTB is caused by Mycobacterium bovis, a member of the M. tuberculosis complex that also includes the aetiological agent for human TB. In this study, we identified a spatio-temporally linked group of 26 cattle and 4 badgers infected with the same Variable Number Tandem Repeat (VNTR) type of M. bovis. Single-nucleotide polymorphisms (SNPs) between sequences identified differences that were consistent with bacterial lineages being persistent on or near farms for several years, despite multiple clear whole herd tests in the interim. Comparing WGS data to mathematical models showed good correlations between genetic divergence and spatial distance, but poor correspondence to the network of cattle movements or within-herd contacts. Badger isolates showed between zero and four SNP differences from the nearest cattle isolate, providing evidence for recent transmissions between the two hosts. This is the first direct genetic evidence of M. bovis persistence on farms over multiple outbreaks with a continued, ongoing interaction with local badgers. However, despite unprecedented resolution, directionality of transmission cannot be inferred at this stage. Despite the often notoriously long timescales between time of infection and time of sampling for TB, our results suggest that WGS data alone can provide insights into TB epidemiology even where detailed contact data are not available, and that more extensive sampling and analysis will allow for quantification of the extent and direction of transmission between cattle and badgers

Genome-wide diversity and phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian dairy cattle

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six “Bison type” isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale

Ions modulate stress-induced nano-texture in supported fluid lipid bilayers.

Most plasma membranes comprise a large number of different molecules including lipids and proteins. In the standard fluid mosaic model, the membrane function is effected by proteins whereas lipids are largely passive and serve solely in the membrane cohesion. Here we show, using supported 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid bilayers in different saline solutions, that ions can locally induce ordering of the lipid molecules within the otherwise fluid bilayer when the latter is supported. This nanoordering exhibits a characteristic length scale of ∼20 nm, and manifests itself clearly when mechanical stress is applied to the membrane. Atomic force microscopy (AFM) measurements in aqueous solutions containing NaCl, KCl, CaCl2, and Tris buffer show that the magnitude of the effect is strongly ion-specific, with Ca2+ and Tris, respectively, promoting and reducing stress-induced nanotexturing of the membrane. The AFM results are complemented by fluorescence recovery after photobleaching experiments, which reveal an inverse correlation between the tendency for molecular nanoordering and the diffusion coefficient within the bilayer. Control AFM experiments on other lipids and at different temperatures support the hypothesis that the nanotexturing is induced by reversible, localized gel-like solidification of the membrane. These results suggest that supported fluid phospholipid bilayers are not homogenous at the nanoscale, but specific ions are able to locally alter molecular organization and mobility, and spatially modulate the membrane’s properties on a length scale of ∼20 nm. To illustrate this point, AFM was used to follow the adsorption of the membrane-penetrating antimicrobial peptide Temporin L in different solutions. The results confirm that the peptides do not absorb randomly, but follow the ion-induced spatial modulation of the membrane. Our results suggest that ionic effects have a significant impact for passively modulating the local properties of biological membranes, when in contact with a support such as the cytoskeleton