The landscape epidemiology of canine rabies virus in Tanzania

Infectious diseases pose a significant threat to animal and human health across the globe, with much of the burden falling on low-income countries. Despite efforts to control many of these diseases, very few have ever been eradicated. Their dynamics are often embedded in complex, heterogeneous landscapes defined by interacting population and landscape level processes. As such, landscape heterogeneity plays a key role in driving disease transmission and persistence. Incorporating landscape heterogeneity in studies of pathogen dynamics is challenging but the accessibility of data, particularly next generation sequencing data, has opened new avenues of research. Landscape epidemiology involves using an integrated approach to understand spatial patterns of disease, using methods that combine landscape genetics, ecology and epidemiology. in this thesis I use these integrative methods to determine the underlying mechanisms facilitating the spread and persistence of canine rabies virus in Tanzania. Whole genome level characterisation of rabies virus samples was achieved and used in combination with cutting-edge inference techniques to explore spatial patterns of rabies at different spatial scales. Phylogeographic patterns were able to characterise spatial scales of endemic rabies transmission in Tanzania, uncovering strong viral population structure at sub-continental levels with evidence of a more fluid dispersal dynamic at local ( less than 100km2 area) spatial scales . Within-country phylogeographic patterns revealed large regional movements within Tanzania that could be attributed to human-mediated movements and revealed the presence of multiple co-circulating lineages within a single administrative district. Finely resolved incidence data from the Serengeti District complemented with whole genome sequences enabled the exploration of local scales of transmission in more detail. By extending phylogeographic diffusion models to incorporate landscape heterogeneity I was able to uncover evidence supporting landscape predictors of rabies diffusion. While much of the spatial structure was attributable to the effects of isolation by distance, landscape predictors had discernible effects on diffusion. In particular, rivers appeared to act as a barrier to dispersal and road networks facilitated diffusion and I found evidence to support vaccination as an effective control measure for canine rabies in the Serengeti District. Importantly, I also found evidence to support vaccination as resistance to diffusion and therefore an effective control measure for dog rabies. As a complementary approach a space-time-genetic algorithm was used to determine who-infected-whom in the Serengeti District. The model explicitly accounted for the possibility of exogenous sources of infection and how to incorporate genetic data available for only a proportion of samples. Direct transmission events were estimated between 42% of observed cases and highlighted the co-circulation of two major lineages in both time and space. Direct transmission events predominantly occurred over very small distances, less than 1km, but a large proportion of cases had unobserved sources that could represent transmission from dogs in neighbouring regions or larger indirect transmission events. A future development of the model is to delineate between these possibilities to assess the true contribution of exogenous sources to the system dynamic. Ultimately these integrative models are at an early stage of development but highlight the power of genetic data to delineate fine-scale transmission patterns. The results from this thesis suggest that landscape features such as rivers could be exploited as barriers in step-wise vaccination campaigns and highlight the utility of genetic surveillance to monitor control and elimination as rabies management progresses

Environmental risk factors for Ixodes ricinus ticks and their infestation on lambs in a changing ecosystem: Implications for tick control and the impact of woodland encroachment on tick-borne disease in livestock

Despite global deforestation some regions, such as Europe, are currently experiencing rapid reforestation. Some of this is unintended woodland encroachment onto farmland as a result of reduced livestock pasture management. Our aim was to determine the likely impacts of this on exposure to ticks and tick-borne disease risk for sheep in Norway, a country experiencing ecosystem changes through rapid woodland encroachment as well as increases in abundance and distribution of Ixodes ricinus ticks and tick-borne disease incidence. We conducted surveys of I. ricinus ticks on ground vegetation using cloth lure transects and counts of ticks biting lambs on spring pastures, where lambs are exposed to infection with Anaplasma phagocytophilum, the causative agent of tick-borne fever in livestock. Pastures had higher densities of I. ricinus ticks on the ground vegetation and more ticks biting lambs if there was more tree cover in or adjacent to pastures. Importantly, there was a close correlation between questing tick density on pastures and counts of ticks biting lambs on the same pasture, indicating that cloth lure transects are a good proxy of risk to livestock of tick exposure and tick-borne disease. These findings can inform policy on environmental tick control measures such as habitat management, choice of livestock grazing area and off-host application of tick control agents

Landscape attributes governing local transmission of an endemic zoonosis: rabies virus in domestic dogs

Landscape heterogeneity plays an important role in disease spread and persistence, but quantifying landscape influences and their scale dependence is challenging. Studies have focused on how environmental features or global transport networks influence pathogen invasion and spread, but their influence on local transmission dynamics that underpin the persistence of endemic diseases remains unexplored. Bayesian phylogeographic frameworks that incorporate spatial heterogeneities are promising tools for analysing linked epidemiological, environmental and genetic data. Here, we extend these methodological approaches to decipher the relative contribu- tion and scale-dependent effects of landscape influences on the transmission of endemic rabies virus in Serengeti district, Tanzania (area ~4,900 km2). Utilizing detailed epidemiological data and 152 complete viral genomes collected between 2004 and 2013, we show that the localized presence of dogs but not their density is the most important determinant of diffusion, implying that culling will be ineffec- tive for rabies control. Rivers and roads acted as barriers and facilitators to viral spread, respectively, and vaccination impeded diffusion despite variable annual cov- erage. Notably, we found that landscape effects were scale-dependent: rivers were barriers and roads facilitators on larger scales, whereas the distribution of dogs was important for rabies dispersal across multiple scales. This nuanced understanding of the spatial processes that underpin rabies transmission can be exploited for targeted control at the scale where it will have the greatest impact. Moreover, this research demonstrates how current phylogeographic frameworks can be adapted to improve our understanding of endemic disease dynamics at different spatial scales

Elucidating the phylodynamics of endemic rabies virus in eastern Africa using whole-genome sequencing

Many of the pathogens perceived to pose the greatest risk to humans are viral zoonoses, responsible for a range of emerging and endemic infectious diseases. Phylogeography is a useful tool to understand the processes that give rise to spatial patterns and drive dynamics in virus populations. Increasingly, whole-genome information is being used to uncover these patterns, but the limits of phylogenetic resolution that can be achieved with this are unclear. Here, whole-genome variation was used to uncover fine-scale population structure in endemic canine rabies virus circulating in Tanzania. This is the first whole-genome population study of rabies virus and the first comprehensive phylogenetic analysis of rabies virus in East Africa, providing important insights into rabies transmission in an endemic system. In addition, sub-continental scale patterns of population structure were identified using partial gene data and used to determine population structure at larger spatial scales in Africa. While rabies virus has a defined spatial structure at large scales, increasingly frequent levels of admixture were observed at regional and local levels. Discrete phylogeographic analysis revealed long-distance dispersal within Tanzania, which could be attributed to human-mediated movement, and we found evidence of multiple persistent, co-circulating lineages at a very local scale in a single district, despite on-going mass dog vaccination campaigns. This may reflect the wider endemic circulation of these lineages over several decades alongside increased admixture due to human-mediated introductions. These data indicate that successful rabies control in Tanzania could be established at a national level, since most dispersal appears to be restricted within the confines of country borders but some coordination with neighbouring countries may be required to limit transboundary movements. Evidence of complex patterns of rabies circulation within Tanzania necessitates the use of whole-genome sequencing to delineate finer scale population structure that can that can guide interventions, such as the spatial scale and design of dog vaccination campaigns and dog movement controls to achieve and maintain freedom from disease

Harnessing technology and portability to conduct molecular epidemiology of endemic pathogens in resource-limited settings

Improvements in genetic and genomic technology have enabled field-deployable molecular laboratories and these have been deployed in a variety of epidemics that capture headlines. In this editorial, we highlight the importance of building physical and personnel capacity in low and middle income countries to deploy these technologies to improve diagnostics, understand transmission dynamics and provide feedback to endemic communities on actionable timelines. We describe our experiences with molecular field research on schistosomiasis, trypanosomiasis and rabies and urge the wider tropical medicine community to embrace these methods and help build capacity to benefit communities affected by endemic infectious diseases

Metabolomics to unveil and understand phenotypic diversity between pathogen populations

Visceral leishmaniasis is caused by a parasite called Leishmania donovani, which every year infects about half a million people and claims several thousand lives. Existing treatments are now becoming less effective due to the emergence of drug resistance. Improving our understanding of the mechanisms used by the parasite to adapt to drugs and achieve resistance is crucial for developing future treatment strategies. Unfortunately, the biological mechanism whereby Leishmania acquires drug resistance is poorly understood. Recent years have brought new technologies with the potential to increase greatly our understanding of drug resistance mechanisms. The latest mass spectrometry techniques allow the metabolome of parasites to be studied rapidly and in great detail. We have applied this approach to determine the metabolome of drug-sensitive and drug-resistant parasites isolated from patients with leishmaniasis. The data show that there are wholesale differences between the isolates and that the membrane composition has been drastically modified in drug-resistant parasites compared with drug-sensitive parasites. Our findings demonstrate that untargeted metabolomics has great potential to identify major metabolic differences between closely related parasite strains and thus should find many applications in distinguishing parasite phenotypes of clinical relevance

Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability

Rapid in-country sequencing of whole virus genomes to inform rabies elimination programmes.

Genomic surveillance is an important aspect of contemporary disease management but has yet to be used routinely to monitor endemic disease transmission and control in low- and middle-income countries. Rabies is an almost invariably fatal viral disease that causes a large public health and economic burden in Asia and Africa, despite being entirely vaccine preventable. With policy efforts now directed towards achieving a global goal of zero dog-mediated human rabies deaths by 2030, establishing effective surveillance tools is critical. Genomic data can provide important and unique insights into rabies spread and persistence that can direct control efforts. However, capacity for genomic research in low- and middle-income countries is held back by limited laboratory infrastructure, cost, supply chains and other logistical challenges. Here we present and validate an end-to-end workflow to facilitate affordable whole genome sequencing for rabies surveillance utilising nanopore technology. We used this workflow in Kenya, Tanzania and the Philippines to generate rabies virus genomes in two to three days, reducing costs to approximately £60 per genome. This is over half the cost of metagenomic sequencing previously conducted for Tanzanian samples, which involved exporting samples to the UK and a three- to six-month lag time. Ongoing optimization of workflows are likely to reduce these costs further. We also present tools to support routine whole genome sequencing and interpretation for genomic surveillance. Moreover, combined with training workshops to empower scientists in-country, we show that local sequencing capacity can be readily established and sustainable, negating the common misperception that cutting-edge genomic research can only be conducted in high resource laboratories. More generally, we argue that the capacity to harness genomic data is a game-changer for endemic disease surveillance and should precipitate a new wave of researchers from low- and middle-income countries