Abstract
. Introduction: Major depressive disorder (MDD) has a significant impact on global burden of disease. Complications in clinical management can occur when response to pharmacological modalities is considered inadequate and symptoms persist (treatment-resistant depression (TRD)). We aim to investigate inflammation, proxied by C-reactive protein (CRP) levels, and body mass index (BMI) as putative causal risk factors for depression and subsequent treatment resistance, leveraging genetic
information to avoid confounding via Mendelian Randomization (MR).

Methods: We used the European UK Biobank subcohort (n = 451, 025), the mental health questionnaire (MHQ) and clinical records. For treatment resistance, a previously curated phenotype based on GP records and prescription data was employed. We applied univariable and multivariable MR models to genetically predict the exposures and assess their causal contribution to a range of depression outcomes. We used weak and pleiotropy, robust estimation techniques within univariable, multivariable and mediation MR models in order to address
our research question with maximum rigour. In addition, we developed a novel statistical procedure to apply pleiotropy robust multivariable MR to one sample data and employed an unfamiliar bootstrap procedure to accurately quantify estimate uncertainty in mediation analysis which outperforms
standard approaches.

Results: in univariable MR models, genetically predicted BMI was positively associated with depression outcomes, including MDD and TRD, with a larger magnitude in women and with age acting
as a moderator of the effect of BMI on PHQ9 (p = 0.011). Multivariable MR analyses suggested an independent causal effect of BMI on TRD not through CRP. Our mediation analyses suggested
that the effect of CRP on PHQ9 was partly mediated by BMI. Individuals with TRD (n = 2, 199) observationally had higher CRP and BMI compared with individuals with MDD alone and healthy
controls. A positive causal association was noted for both the exposures, but in multivariable analyses only BMI retained statistical significance at the 5% level.

Discussion: Our work supportst the assertion BMI exerts a causal effect on a range of clinical and questionnaire-based depression phenotypes, with the effect being stronger in women and in younger individuals. We show that this effect is independent of inflammation proxied by CRP levels as the effects of CRP do not persist when jointly estimated with BMI. This is consistent with previous evidence suggesting that overweight contributed to depression even in the absence of any metabolic consequences. It appears that BMI exerts an effect on TRD that persists when we account for BMI influencing MDD.

AbstractThe emergence and wide-spread circulation of mosquito-transmitted viral diseases, such as dengue, Zika and Chikungunya, is a global public health concern. In the absence of effective vaccines, current control measures are mostly targeted against the mosquito vector and have so far only shown limited success. The reliance on mosquitoes for transmission also imposes strong ecological constraints that can introduce significant spatial and temporal variations in disease incidence. However, the way that epidemiological and ecological factors interact and determine population-level disease dynamics is only partially understood. Here we fit a spatially-explicit individual based model defined within a Bayesian framework to Zika incidence data from Feira de Santana, allowing us to more precisely quantify the relationships between socio-ecological factors and arboviral outbreaks. Our results further demonstrated that the virus was likely introduced into multiple spatially segregated locations at the start of the outbreak, highlighting the benefits that spatio-temporal incidence data would bring in making modelling approaches more realistic for public health planning.

AbstractA precision medicine approach in type 2 diabetes (T2D) could enhance targeting specific glucose-lowering therapies to individual patients most likely to benefit. We utilised Bayesian non-parametric modelling to develop and validate an individualised treatment selection algorithm for two major T2D drug classes, SGLT2-inhibitors (SGLT2i) and GLP1-receptor agonists (GLP1-RA). The algorithm is designed to predict differences in 12-month glycaemic outcome (HbA1c) between the 2 therapies, based on routine clinical features of 46,394 people with T2D in England (27,319 for model development, 19,075 for hold-out validation), with additional external validation in 2,252 people with T2D from Scotland. Routine clinical features, including sex (with females markedly more responsive to GLP1-RA), were associated with differences in glycaemic outcomes. Our algorithm identifies clearly delineable subgroups with reproducible ≥5mmol/mol HbA1cbenefits associated with each drug class. Moreover, we demonstrate that targeting the therapies based on predicted glycaemic response is associated with improvements in short-term tolerability and long-term risk of new-onset microvascular complications. These results show that precision medicine approaches to T2D can facilitate effective individualised treatment selection, and that use of routinely collected clinical features could support low-cost deployment in many countries.

Infectious disease models are widely used by epidemiologists to improve the understanding of transmission dynamics and disease natural history, and to predict the possible effects of interventions. As the complexity of such models increases, however, it becomes increasingly challenging to robustly calibrate them to empirical data. History matching with emulation is a calibration method that has been successfully applied to such models, but has not been widely used in epidemiology partly due to the lack of available software. To address this issue, we developed a new, user-friendly R package hmer to simply and efficiently perform history matching with emulation. In this paper, we demonstrate the first use of hmer for calibrating a complex deterministic model for the country-level implementation of tuberculosis vaccines to 115 low- and middle-income countries. The model was fit to 9-13 target measures, by varying 19-22 input parameters. Overall, 105 countries were successfully calibrated. Among the remaining countries, hmer visualisation tools, combined with derivative emulation methods, provided strong evidence that the models were misspecified and could not be calibrated to the target ranges. This work shows that hmer can be used to simply and rapidly calibrate a complex model to data from over 100 countries, making it a useful addition to the epidemiologist's calibration tool-kit.

Abstract


Genome‐wide homozygosity, caused for example by inbreeding, is expected to have deleterious effects on survival and/or reproduction. Evolutionary theory predicts that any fitness costs are likely to be detected in late life because natural selection will filter out negative impacts on younger individuals with greater reproductive value.

Here we infer associations between multi‐locus homozygosity (MLH), sex, disease and age‐dependent mortality risks using Bayesian analysis of the life histories of wild European badgers Meles meles in a population naturally infected with Mycobacterium bovis (the causative agent of bovine tuberculosis [bTB]).

We find important effects of MLH on all parameters of the Gompertz–Makeham mortality hazard function, but particularly in later life.

Our findings confirm the predicted association between genomic homozygosity and actuarial senescence. Increased homozygosity is particularly associated with an earlier onset, and greater rates of actuarial senescence, regardless of sex. The association between homozygosity and actuarial senescence is further amplified among badgers putatively infected with bTB.

These results recommend further investigation into the ecological and behavioural processes that result in genome‐wide homozygosity, and focused work on whether homozygosity is harmful or beneficial during early life‐stages.

Global health is becoming increasingly reliant on our understanding and management of wildlife disease. An estimated 60% of emerging infectious diseases in humans are zoonotic and with human-wildlife interactions set to increase as populations rise and we expand further into wild habitats there is pressure to seek modelling frameworks that enable a deeper understanding of natural systems.
Survival and mortality are fundamental parameters of interest when investigating the impact of disease with far reaching implications for species conservation, management and control. Survival analysis has traditionally been dominated by non- and semi-parametric methods but these can sometimes miss subtle yet important dynamics. Survival and mortality trajectory analysis can alleviate some of these problems by fitting fully parametric functions that describe lifespan patterns of mortality and survival. In the first part of this thesis we investigate the use of survival and mortality trajectories in epidemiology and uncover novel patterns of age-, sex- and infection-specific mortality in a wild population of European badgers (Meles meles) naturally infected with Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB). Limitations of dedicated software packages to conduct such analyses led us to investigate alternative methods to build models from first principles and we found the NIMBLE package to offer an attractive blend of flexibility and speed. We create a novel parameterisation of the Siler model to enable more flexible model specification but encounter the common problem of competing models having comparable fits to the data. Multi-model inference approaches can alleviate some of these issues but require efficient methods to carry out model comparisons; we present an approach based on the estimation of the marginal likelihood through importance sampling and demonstrate its application through a series of simulation- and case-studies. The approach works well for both census and capture-mark-recapture (CMR) data, both of which are common within ecological research, but we uncover challenges in recording and modelling early life mortality dynamics that occur as a result of the CMR sampling process. The final part of the thesis looks at another alternative approach for model comparison that doesn’t require direct estimation of the marginal likelihood, Reversible Jump Markov Chain Monte Carlo (RJMCMC), which is particularly efficient when models to be compared are nested and the problem can reduce to one of variable selection. In the final chapter we carry out an investigation of age-, sex-, infection- and inbreeding-specific variation in survival and mortality in a wild population of European badgers naturally infected with bovine Tuberculosis. Using the methods and knowledge presented through the earlier chapters of this thesis we uncover patterns of mortality consistent with both the mutation accumulation and antagonistic pleiotropy theories of senescence but most interestingly uncover antagonistic pleiotropic effects of inbreeding on age-specific mortality in a wild population for the first time.
This thesis provides a number of straightforward approaches to Bayesian survival analysis that are widely applicable to ecological research and can offer greater insight and uncover subtle patterns of survival and mortality that traditional methods can overlook. Our investigation into the epidemiology of bovine Tuberculosis and in particular the effects of inbreeding have far-reaching implications for the control of this disease. This research can also inform future conservation efforts and management strategies as all species are likely to be at increasing risk of inbreeding in an age of dramatic global change, rapid habitat loss and isolation.

CONTEXT: the importance of the autoantibody level at diagnosis of type 1 diabetes (T1D) is not clear. OBJECTIVE: We aimed to assess the association of glutamate decarboxylase (GADA), islet antigen-2 (IA-2A), and zinc transporter 8 (ZnT8A) autoantibody levels with clinical and genetic characteristics at diagnosis of T1D. METHODS: We conducted a prospective, cross-sectional study. GADA, IA-2A, and ZnT8A were measured in 1644 individuals with T1D at diagnosis using radiobinding assays. Associations between autoantibody levels and the clinical and genetic characteristics for individuals were assessed in those positive for these autoantibodies. We performed replication in an independent cohort of 449 people with T1D. RESULTS: GADA and IA-2A levels exhibited a bimodal distribution at diagnosis. High GADA level was associated with older age at diagnosis (median 27 years vs 19 years, P = 9 × 10-17), female sex (52% vs 37%, P = 1 × 10-8), other autoimmune diseases (13% vs 6%, P = 3 × 10-6), and HLA-DR3-DQ2 (58% vs 51%, P =. 006). High IA-2A level was associated with younger age of diagnosis (median 17 years vs 23 years, P = 3 × 10-7), HLA-DR4-DQ8 (66% vs 50%, P = 1 × 10-6), and ZnT8A positivity (77% vs 52%, P = 1 × 10-15). We replicated our findings in an independent cohort of 449 people with T1D where autoantibodies were measured using enzyme-linked immunosorbent assays. CONCLUSION: Islet autoantibody levels provide additional information over positivity in T1D at diagnosis. Bimodality of GADA and IA-2A autoantibody levels highlights the novel aspect of heterogeneity of T1D. This may have implications for T1D prediction, treatment, and pathogenesis.

BACKGROUND: Variation in host attributes that influence their contact rates and infectiousness can lead some individuals to make disproportionate contributions to the spread of infections. Understanding the roles of such 'superspreaders' can be crucial in deciding where to direct disease surveillance and controls to greatest effect. In the epidemiology of bovine tuberculosis (bTB) in Great Britain, it has been suggested that a minority of cattle farms or herds might make disproportionate contributions to the spread of Mycobacterium bovis, and hence might be considered 'superspreader farms'. OBJECTIVES AND METHODS: We review the literature to identify the characteristics of farms that have the potential to contribute to exceptional values in the three main components of the farm reproductive number - Rf : contact rate, infectiousness and duration of infectiousness, and therefore might characterize potential superspreader farms for bovine tuberculosis in Great Britain. RESULTS: Farms exhibit marked heterogeneity in contact rates arising from between-farm trading of cattle. A minority of farms act as trading hubs that greatly augment connections within cattle trading networks. Herd infectiousness might be increased by high within-herd transmission or the presence of supershedding individuals, or infectiousness might be prolonged due to undetected infections or by repeated local transmission, via wildlife or fomites. CONCLUSIONS: Targeting control methods on putative superspreader farms might yield disproportionate benefits in controlling endemic bovine tuberculosis in Great Britain. However, real-time identification of any such farms, and integration of controls with industry practices, present analytical, operational and policy challenges.

Trading animals between farms and via markets can provide a conduit for spread of infections. By studying trading networks, we might better understand the dynamics of livestock diseases. We constructed ingoing contact chains of cattle farms in Great Britain that were linked by trading, to elucidate potential pathways for the transmission of infection and to evaluate their effect on the risk of a farm experiencing a bovine tuberculosis (bTB) incident. Our findings are consistent with variation in bTB risk associated with region, herd size, disease risk area and history of previous bTB incidents on the root farm and nearby farms. However, we also identified effects of both direct and indirect trading patterns, such that connections to more farms in the England High-Risk Area up to three movements away from the root farm increased the odds of a bTB incident, while connections with more farms in the England Low-Risk Area up to eight movements away decreased the odds. Relative to other risk factors for bTB, trading behaviours are arguably more amenable to change, and consideration of risks associated with indirect trading, as well direct trading, might therefore offer an additional approach to bTB control in Great Britain.

Combinations of intense non-pharmaceutical interventions ('lockdowns') were
introduced in countries worldwide to reduce SARS-CoV-2 transmission. Many
governments have begun to implement lockdown exit strategies that allow
restrictions to be relaxed while attempting to control the risk of a surge in
cases. Mathematical modelling has played a central role in guiding
interventions, but the challenge of designing optimal exit strategies in the
face of ongoing transmission is unprecedented. Here, we report discussions from
the Isaac Newton Institute 'Models for an exit strategy' workshop (11-15 May
2020). A diverse community of modellers who are providing evidence to
governments worldwide were asked to identify the main questions that, if
answered, will allow for more accurate predictions of the effects of different
exit strategies. Based on these questions, we propose a roadmap to facilitate
the development of reliable models to guide exit strategies. The roadmap
requires a global collaborative effort from the scientific community and
policy-makers, and is made up of three parts: i) improve estimation of key
epidemiological parameters; ii) understand sources of heterogeneity in
populations; iii) focus on requirements for data collection, particularly in
Low-to-Middle-Income countries. This will provide important information for
planning exit strategies that balance socio-economic benefits with public
health.

Infectious diseases of livestock can cause substantial production losses and have detrimental impacts upon human health, and animal health and welfare. To limit the impact of diseases, understanding more about the dynamics of transmission can assist in the control and prevention of infectious disease. In particular, understanding infection transmission on networks, ‘network epidemiology’, offers a flexible approach, incorporating between-host heterogeneity in potentially infectious contacts drawn from empirical study of interactions among individual animals, or among farms. Trading animals and optimising productivity are vital to the commercial viability of farms, however they necessarily involve compromises in biosecurity, animal health, and welfare. Better understanding of the relationships among these multiple factors might facilitate the development of sustainable livestock industries that are more resilient to disease outbreaks.
In this thesis I examine cattle interactions at two spatial scales, first at a national-level by studying the trading connections among farms, and then at a finer scale by analysing the social interactions among cattle. First, I introduce the concept of superspreaders, hosts that generate many more secondary infections than the rest of the population, and evaluate evidence for the notion that some farms might act as superspreaders of infection. I utilise the example of bovine tuberculosis (bTB) to illustrate this concept and find that farms might act as superspreaders in three main ways; first, via exceptional trading between farms, second, by factors that facilitate high within-herd transmission and trading of high-risk animals, and third, by harbouring undetected infection for long periods. I find mechanisms that align with all three processes in the cattle industry in Great Britain that might allow superspreader farms to contribute to the current bTB epidemic.
At a national level, I describe cattle movements among farms over time, finding that some farms consistently act as ‘hubs’ in trading networks, functioning in a similar way to markets, in that they are highly connected to other farms by many direct trades. Utilising the temporal network measure of ‘contact chains’, I quantify the farms that represent potential sources of infection (ingoing contact chains) and the potential farms that a farm might infect over 1 year periods. Farms divide into two groups: those with very few connections (less than 10 farms) that are relatively isolated from the network, and those with very many connections (more than 1000 farms) that are highly connected within the network. I find that a substantial number of farms have over 10,000 farms in both their ingoing and outgoing contact chains, such that, if infected, they might potentially act as superspreaders by being more at risk of both acquiring and spreading infection.
Building on my previous analysis, I then characterise the ‘source farms’ in the ingoing contact chains, in terms of their location and bTB history. I find that after controlling for previously-established risk factors for bTB, having more source farms in areas of higher bTB risk in the ingoing contact chain increases the odds of a bTB incident on the root farm, whilst having more source farms in lower risk areas is associated with lower odds of a bTB incident on the root farm.
At a finer scale of contacts among animals, I explore interactions among dairy cattle in multiple herds using automated proximity sensors and GPS devices. When aggregated over long periods, cattle interactions appear dense and unstructured, however finer time spatial and temporal perspectives revealed structure and variation in contacts. Herds in our study had variable grazing and housing access, allowing us to determine that cattle interact with more other cows, for longer time periods when they are in buildings compared to contacts at pasture. Cattle exhibited heterogeneity in their number and duration of contacts, and although the majority of cattle interacted more equally with other cows, a small proportion of cows in each group showed evidence of stronger social ties.
Next, I consider associations between social interactions, production, and health. I review the existing literature on social parameters such as dominance rank and re-grouping of cattle, and find inconclusive outcomes regarding their impact on milk yield and somatic cell count, an indicator of udder health. I perform my own analysis to examine the relationship between the time cows spend with other cows, milk yield and somatic cell count, and do not find a statistically significant relationship. In considering social preference, cows that had experienced the same number of lactations were more likely to interact, but cows spending more time with cows in the same lactation did not appreciably affect their milk yield or somatic cell count.
Finally, I draw together the findings of this thesis and reflect on how the identification of higher-risk farms might be useful in the control of livestock infections, and specifically bTB in Great Britain. I conclude that network analysis is a valuable tool to study the interactions of cattle and cattle farms, identifying unique opportunities for targeted approaches to disease control.

The dengue virus (DENV) remains a considerable global public health concern. The interactions between the virus, its mosquito vectors and the human host are complex and only partially understood. Dependencies of vector ecology on environmental attributes, such as temperature and rainfall, together with host population density, introduce strong spatiotemporal heterogeneities, resulting in irregular epidemic outbreaks and asynchronous oscillations in serotype prevalence. Human movements across different spatial scales have also been implicated as important drivers of dengue epidemiology across space and time, and further create the conditions for the geographic expansion of dengue into new habitats. Previously proposed transmission models often relied on strong, unrealistic assumptions regarding key epidemiological and ecological interactions to elucidate the effects of these spatio-temporal heterogeneities on the emergence, spread and persistence of dengue. Furthermore, the computational limitations of individual based models have hindered the development of more detailed descriptions of the influence of vector ecology, environment and human mobility on dengue epidemiology.

In order to address these shortcomings, the main aim of this thesis was to rigorously quantify the effects of ecological drivers on dengue epidemiology within a robust and computational efficient framework. The individual based model presented included an explicit spatial structure, vector and human movement, spatio-temporal heterogeneity in population densities, and climate effects. The flexibility of the framework allowed robust assessment of the implications of classical modelling assumptions on the basic reproduction number, R₀, demonstrating that traditional approaches grossly inflate R₀ estimates. The model's more realistic meta-population formulation was then exploited to elucidate the effects of ecological heterogeneities on dengue incidence which showed that sufficient levels of community connectivity are required for the spread and persistence of dengue virus. By fitting the individual based model to empirical data, the influence of climate and on dengue was quantified, revealing the strong benefits that cross-sectional serological data could bring to more precisely inferring ecological drivers of arboviral epidemiology. Overall, the findings presented here demonstrate the wide epidemiological landscape which ecological drivers induce, forewarning against the strong implications of generalising interpretations from one particular setting across wider spatial contexts. These findings will prove invaluable for the assessment of vector-borne control strategies, such as mosquito elimination or vaccination deployment programs.

BACKGROUND: Uganda changed its antiretroviral therapy guidelines in 2014, increasing the CD4 threshold for antiretroviral therapy initiation from 350 cells/μl to 500 cells/μl. We investigate what effect this change in policy is likely to have on HIV incidence, morbidity, and programme costs, and estimate the cost-effectiveness of the change over different time horizons. METHODS: We used a complex individual-based model of HIV transmission and antiretroviral therapy scale-up in Uganda. 100 model fits were generated by fitting the model to 51 demographic, sexual behaviour, and epidemiological calibration targets, varying 96 input parameters, using history matching with model emulation. An additional 19 cost and disability weight parameters were varied during the analysis of the model results. For each model fit, the model was run to 2030, with and without the change in threshold to 500 cells/μl. RESULTS: the change in threshold led to a 9.7% (90% plausible range: 4.3%-15.0%) reduction in incidence in 2030, and averted 278,944 (118,452-502,790) DALYs, at a total cost of $28M (-$142M to +$195M). The cost per disability adjusted life year (DALY) averted fell over time, from $3238 (-$125 to +$29,969) in 2014 to $100 (-$499 to +$785) in 2030. The change in threshold was cost-effective (cost

BACKGROUND: UNAIDS calls for fewer than 500,000 new HIV infections/year by 2020, with treatment-as-prevention being a key part of their strategy for achieving the target. A better understanding of the contribution to transmission of people at different stages of the care pathway can help focus intervention services at populations where they may have the greatest effect. We investigate this using Uganda as a case study. METHODS: an individual-based HIV/ART model was fitted using history matching. 100 model fits were generated to account for uncertainties in sexual behaviour, HIV epidemiology, and ART coverage up to 2015 in Uganda. A number of different ART scale-up intervention scenarios were simulated between 2016 and 2030. The incidence and proportion of transmission over time from people with primary infection, post-primary ART-naïve infection, and people currently or previously on ART was calculated. RESULTS: in all scenarios, the proportion of transmission by ART-naïve people decreases, from 70% (61%-79%) in 2015 to between 23% (15%-40%) and 47% (35%-61%) in 2030. The proportion of transmission by people on ART increases from 7.8% (3.5%-13%) to between 14% (7.0%-24%) and 38% (21%-55%). The proportion of transmission by ART dropouts increases from 22% (15%-33%) to between 31% (23%-43%) and 56% (43%-70%). CONCLUSIONS: People who are currently or previously on ART are likely to play an increasingly large role in transmission as ART coverage increases in Uganda. Improving retention on ART, and ensuring that people on ART remain virally suppressed, will be key in reducing HIV incidence in Uganda.

Campylobacter jejuni, the most common cause of bacterial diarrhoeal disease, is normally helical. However, it can also adopt straight rod, elongated helical and coccoid forms. Studying how helical morphology is generated, and how it switches between its different forms, is an important objective for understanding this pathogen. Here, we aimed to determine the genetic factors involved in generating the helical shape of Campylobacter. A C. jejuni transposon (Tn) mutant library was screened for non-helical mutants with inconsistent results. Whole genome sequence variation and morphological trends within this Tn library, and in various C. jejuni wild type strains, were compared and correlated to detect genomic elements associated with helical and rod morphologies. All rod-shaped C. jejuni Tn mutants and all rod-shaped laboratory, clinical and environmental C. jejuni and Campylobacter coli contained genetic changes within the pgp1 or pgp2 genes, which encode peptidoglycan modifying enzymes. We therefore confirm the importance of Pgp1 and Pgp2 in the maintenance of helical shape and extended this to a wide range of C. jejuni and C. coli isolates. Genome sequence analysis revealed variation in the sequence and length of homopolymeric tracts found within these genes, providing a potential mechanism of phase variation of cell shape.

Advances in scientific computing have allowed the development of complex models that are being routinely applied to problems in disease epidemiology, public health and decision making. The utility of these models depends in part on how well they can reproduce empirical data. However, fitting such models to real world data is greatly hindered both by large numbers of input and output parameters, and by long run times, such that many modelling studies lack a formal calibration methodology. We present a novel method that has the potential to improve the calibration of complex infectious disease models (hereafter called simulators). We present this in the form of a tutorial and a case study where we history match a dynamic, event-driven, individual-based stochastic HIV simulator, using extensive demographic, behavioural and epidemiological data available from Uganda. The tutorial describes history matching and emulation. History matching is an iterative procedure that reduces the simulator's input space by identifying and discarding areas that are unlikely to provide a good match to the empirical data. History matching relies on the computational efficiency of a Bayesian representation of the simulator, known as an emulator. Emulators mimic the simulator's behaviour, but are often several orders of magnitude faster to evaluate. In the case study, we use a 22 input simulator, fitting its 18 outputs simultaneously. After 9 iterations of history matching, a non-implausible region of the simulator input space was identified that was 10(11) times smaller than the original input space. Simulator evaluations made within this region were found to have a 65% probability of fitting all 18 outputs. History matching and emulation are useful additions to the toolbox of infectious disease modellers. Further research is required to explicitly address the stochastic nature of the simulator as well as to account for correlations between outputs.

Vaccination for the control of bovine tuberculosis (bTB) in cattle is not currently used within any international control program, and is illegal within the EU. Candidate vaccines, based upon Mycobacterium bovis bacillus Calmette-Guérin (BCG) all interfere with the action of the tuberculin skin test, which is used to determine if animals, herds and countries are officially bTB-free. New diagnostic tests that Differentiate Infected from Vaccinated Animals (DIVA) offer the potential to introduce vaccination within existing eradication programs. We use within-herd transmission models estimated from historical data from Great Britain (GB) to explore the feasibility of such supplemental use of vaccination. The economic impact of bovine Tuberculosis for farmers is dominated by the costs associated with testing, and associated restrictions on animal movements. Farmers' willingness to adopt vaccination will require vaccination to not only reduce the burden of infection, but also the risk of restrictions being imposed. We find that, under the intensive sequence of testing in GB, it is the specificity of the DIVA test, rather than the sensitivity, that is the greatest barrier to see a herd level benefit of vaccination. The potential negative effects of vaccination could be mitigated through relaxation of testing. However, this could potentially increase the hidden burden of infection within Officially TB Free herds. Using our models, we explore the range of the DIVA test characteristics necessary to see a protective herd level benefit of vaccination. We estimate that a DIVA specificity of at least 99.85% and sensitivity of >40% is required to see a protective benefit of vaccination with no increase in the risk of missed infection. Data from experimentally infected animals suggest that this target specificity could be achieved in vaccinates using a cocktail of three DIVA antigens while maintaining a sensitivity of 73.3% (95%CI: 61.9, 82.9%) relative to post-mortem detection.

Canine rabies can be effectively controlled by vaccination with readily available, high-quality vaccines. These vaccines should provide protection from challenge in healthy dogs, for the claimed period, for duration of immunity, which is often two or three years. It has been suggested that, in free-roaming dog populations where rabies is endemic, vaccine-induced protection may be compromised by immuno-suppression through malnutrition, infection and other stressors. This may reduce the proportion of dogs that seroconvert to the vaccine during vaccination campaigns and the duration of immunity of those dogs that seroconvert. Vaccination coverage may also be limited through insufficient vaccine delivery during vaccination campaigns and the loss of vaccinated individuals from populations through demographic processes. This is the first longitudinal study to evaluate temporal variations in rabies vaccine-induced serological responses, and factors associated with these variations, at the individual level in previously unvaccinated free-roaming dog populations. Individual-level serological and health-based data were collected from three cohorts of dogs in regions where rabies is endemic, one in South Africa and two in Indonesia. We found that the vast majority of dogs seroconverted to the vaccine; however, there was considerable variation in titres, partly attributable to illness and lactation at the time of vaccination. Furthermore, >70% of the dogs were vaccinated through community engagement and door-to-door vaccine delivery, even in Indonesia where the majority of the dogs needed to be caught by net on successive occasions for repeat blood sampling and vaccination. This demonstrates the feasibility of achieving population-level immunity in free-roaming dog populations in rabies-endemic regions. However, attrition of immune individuals through demographic processes and waning immunity necessitates repeat vaccination of populations within at least two years to ensure communities are protected from rabies. These findings support annual mass vaccination campaigns as the most effective means to control canine rabies.

A powerful and flexible method for fitting dynamic models to missing and censored data is to use the Bayesian paradigm via data-augmented Markov chain Monte Carlo (DA-MCMC). This samples from the joint posterior for the parameters and missing data, but requires high memory overheads for large-scale systems. In addition, designing efficient proposal distributions for the missing data is typically challenging. Pseudo-marginal methods instead integrate across the missing data using a Monte Carlo estimate for the likelihood, generated from multiple independent simulations from the model. These techniques can avoid the high memory requirements of DA-MCMC, and under certain conditions produce the exact marginal posterior distribution for parameters. A novel method is presented for implementing importance sampling for dynamic epidemic models, by conditioning the simulations on sets of validity criteria (based on the model structure) as well as the observed data. The flexibility of these techniques is illustrated using both removal time and final size data from an outbreak of smallpox. It is shown that these approaches can circumvent the need for reversible-jump MCMC, and can allow inference in situations where DA-MCMC is impossible due to computationally infeasible likelihoods. © 2013 Elsevier B.V. All rights reserved.

Summary: Understanding the demography of domestic dog populations is essential for effective disease control, particularly of canine-mediated rabies. Demographic data are also needed to plan effective population management. However, no study has comprehensively evaluated the contribution of demographic processes (i.e. births, deaths and movement) to variations in dog population size or density, or determined the factors that regulate these processes, including human factors. We report the results of a 3-year cohort study of domestic dogs, which is the first to generate detailed data on the temporal variation of these demographic characteristics. The study was undertaken in two communities in each of Bali, Indonesia and Johannesburg, South Africa, in rabies-endemic areas and where the majority of dogs were free-roaming. None of the four communities had been engaged in any dog population management interventions by local authorities or animal welfare organizations. All identified dogs in the four communities were monitored individually throughout the study. We observed either no population growth or a progressive decline in population size during the study period. There was no clear evidence that population size was regulated through environmental resource constraints. Rather, almost all of the identified dogs were owned and fed regularly by their owners, consistent with population size regulated by human demand. Finally, a substantial fraction of the dogs originated from outside the population, entirely through the translocation of dogs by people, rather than from local births. These findings demonstrate that previously reported growth of dog populations is not a general phenomenon and challenge the widely held view that free-roaming dogs are unowned and form closed populations. Synthesis and applications. These observations have broad implications for disease and population control. The accessibility of dogs for vaccination and evaluation through owners and the movement of dogs (some of them infected) by people will determine the viable options for disease control strategies. The impact of human factors on population dynamics will also influence the feasibility of annual vaccination campaigns to control rabies and population control through culling or sterilization. The complex relationship between dogs and people is critically important in the transmission and control of canine-mediated rabies. For effective management, human factors must be considered in the development of disease and population control programmes. © 2014 the Authors Journal of Applied Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society.

Bovine tuberculosis (BTB) is an important livestock disease, seriously impacting cattle industries in both industrialised and pre-industrialised countries. Like TB in other mammals, infection is life long and, if undiagnosed, may progress to disease years after exposure. The risk of disease in humans is highly age-dependent, however in cattle, age-dependent risks have yet to be quantified, largely due to insufficient data and limited diagnostics. Here, we estimate age-specific reactor rates in Great Britain by combining herd-level testing data with spatial movement data from the Cattle Tracing System (CTS). Using a catalytic model, we find strong age dependencies in infection risk and that the probability of detecting infection increases with age. Between 2004 and 2009, infection incidence in cattle fluctuated around 1%. Age-specific incidence increased monotonically until 24-36 months, with cattle aged between 12 and 36 months experiencing the highest rates of infection. Beef and dairy cattle under 24 months experienced similar infection risks, however major differences occurred in older ages. The average reproductive number in cattle was greater than 1 for the years 2004-2009. These methods reveal a consistent pattern of BTB rates with age, across different population structures and testing patterns. The results provide practical insights into BTB epidemiology and control, suggesting that targeting a mass control programme at cattle between 12 and 36 months could be beneficial.

Understanding the genetic, antigenic and structural changes that occur during HIV-1 infection in response to pre-existing immunity will facilitate current efforts to develop an HIV-1 vaccine. Much is known about HIV-1 variation at the population level but little with regard to specific changes occurring in the envelope glycoprotein within a host in response to immune pressure elicited by antibodies. The aim of this study was to track and map specific early genetic changes occurring in the viral envelope gene following vaccination using a highly controlled viral challenge setting in the SHIV macaque model. We generated 449 full-length env sequences from vaccinees, and 63 from the virus inoculum. Analysis revealed a different pattern in the distribution and frequency of mutations in the regions of the envelope gene targeted by the vaccine as well as different patterns of diversification between animals in the naïve control group and vaccinees. Given the high stringency of the model it is remarkable that we were able to identify genetic changes associated with the vaccination. This work provides insight into the characterization of breakthrough viral populations in less than fully efficacious vaccines and illustrates the value of HIV-1 Env SHIV challenge model in macaques to unravel the mechanisms driving HIV-1 envelope genetic diversity in the presence of vaccine induced-responses.

Understanding the mechanisms underlying the observed dynamics of complex biological systems requires the statistical assessment and comparison of multiple alternative models. Although this has traditionally been done using maximum likelihood-based methods such as Akaike's Information Criterion (AIC), Bayesian methods have gained in popularity because they provide more informative output in the form of posterior probability distributions. However, comparison between multiple models in a Bayesian framework is made difficult by the computational cost of numerical integration over large parameter spaces. A new, efficient method for the computation of posterior probabilities has recently been proposed and applied to complex problems from the physical sciences. Here we demonstrate how nested sampling can be used for inference and model comparison in biological sciences. We present a reanalysis of data from experimental infection of mice with Salmonella enterica showing the distribution of bacteria in liver cells. In addition to confirming the main finding of the original analysis, which relied on AIC, our approach provides: (a) integration across the parameter space, (b) estimation of the posterior parameter distributions (with visualisations of parameter correlations), and (c) estimation of the posterior predictive distributions for goodness-of-fit assessments of the models. The goodness-of-fit results suggest that alternative mechanistic models and a relaxation of the quasi-stationary assumption should be considered.

Antibodies are known to be essential in controlling Salmonella infection, but their exact role remains elusive. We recently developed an in vitro model to investigate the relative efficiency of four different human immunoglobulin G (IgG) subclasses in modulating the interaction of the bacteria with human phagocytes. Our results indicated that different IgG subclasses affect the efficacy of Salmonella uptake by human phagocytes. In this study, we aim to quantify the effects of IgG on intracellular dynamics of infection by combining distributions of bacterial numbers per phagocyte observed by fluorescence microscopy with a mathematical model that simulates the in vitro dynamics. We then use maximum likelihood to estimate the model parameters and compare them across IgG subclasses. The analysis reveals heterogeneity in the division rates of the bacteria, strongly suggesting that a subpopulation of intracellular Salmonella, while visible under the microscope, is not dividing. Clear differences in the observed distributions among the four IgG subclasses are best explained by variations in phagocytosis and intracellular dynamics. We propose and compare potential factors affecting the replication and death of bacteria within phagocytes, and we discuss these results in the light of recent findings on dormancy of Salmonella.

Reservoir hosts of novel pathogens are often identified or suspected as such on the basis of serological assay results, prior to the isolation of the pathogen itself. Serological assays might therefore be used outside of their original, validated scope in order to infer seroprevalences in reservoir host populations, until such time that specific diagnostic assays can be developed. This is particularly the case in wildlife disease research. The absence of positive and negative control samples and gold standard diagnostic assays presents challenges in determining an appropriate threshold, or 'cutoff', for the assay that enables differentiation between seronegative and seropositive individuals. Here, multiple methods were explored to determine an appropriate cutoff for a multiplexed microsphere assay that is used to detect henipavirus antibody binding in fruit bat plasma. These methods included calculating multiples of 'negative' control assay values, receiver operating characteristic curve analyses, and Bayesian mixture models to assess the distribution of assay outputs for classifying seropositive and seronegative individuals within different age classes. As for any diagnostic assay, the most appropriate cutoff determination method and value selected must be made according to the aims of the study. This study is presented as an example for others where reference samples, and assays that have been characterised previously, are absent.

Intracellular replication within specialized vacuoles and cell-to-cell spread in the tissue are essential for the virulence of Salmonella enterica. By observing infection dynamics at the single-cell level in vivo, we have discovered that the Salmonella pathogenicity island 2 (SPI-2) type 3 secretory system (T3SS) is dispensable for growth to high intracellular densities. This challenges the concept that intracellular replication absolutely requires proteins delivered by SPI-2 T3SS, which has been derived largely by inference from in vitro cell experiments and from unrefined measurement of net growth in mouse organs. Furthermore, we infer from our data that the SPI-2 T3SS mediates exit from infected cells, with consequent formation of new infection foci resulting in bacterial spread in the tissues. This suggests a new role for SPI-2 in vivo as a mediator of bacterial spread in the body. In addition, we demonstrate that very similar net growth rates of attenuated salmonellae in organs can be derived from very different underlying intracellular growth dynamics.

Salmonella enterica causes a range of diseases. Salmonellae are intracellular parasites of macrophages, and the control of bacteria within these cells is critical to surviving an infection. The dynamics of the bacteria invading, surviving, proliferating in and killing macrophages are central to disease pathogenesis. Fundamentally important parameters, however, such as the cellular infection rate, have not previously been calculated. We used two independent approaches to calculate the macrophage infection rate: mathematical modelling of Salmonella infection experiments, and analysis of real-time video microscopy of infection events. Cells repeatedly encounter salmonellae, with the bacteria often remain associated with the macrophage for more than ten seconds. Once Salmonella encounters a macrophage, the probability of that bacterium infecting the cell is remarkably low: less than 5%. The macrophage population is heterogeneous in terms of its susceptibility to the first infection event. Once infected, a macrophage can undergo further infection events, but these reinfection events occur at a lower rate than that of the primary infection.

The number of cattle herds placed under movement restrictions in Great Britain (GB) due to the suspected presence of bovine tuberculosis (bTB) has progressively increased over the past 25 years despite an intensive and costly test-and-slaughter control program. Around 38% of herds that clear movement restrictions experience a recurrent incident (breakdown) within 24 months, suggesting that infection may be persisting within herds. Reactivity to tuberculin, the basis of diagnostic testing, is dependent on the time from infection. Thus, testing efficiency varies between outbreaks, depending on weight of transmission and cannot be directly estimated. In this paper, we use Approximate Bayesian Computation (ABC) to parameterize two within-herd transmission models within a rigorous inferential framework. Previous within-herd models of bTB have relied on ad-hoc methods of parameterization and used a single model structure (SORI) where animals are assumed to become detectable by testing before they become infectious. We study such a conventional within-herd model of bTB and an alternative model, motivated by recent animal challenge studies, where there is no period of epidemiological latency before animals become infectious (SOR). Under both models we estimate that cattle-to-cattle transmission rates are non-linearly density dependent. The basic reproductive ratio for our conventional within-herd model, estimated for scenarios with no statutory controls, increases from 1.5 (0.26-4.9; 95% CI) in a herd of 30 cattle up to 4.9 (0.99-14.0) in a herd of 400. Under this model we estimate that 50% (33-67) of recurrent breakdowns in Britain can be attributed to infection missed by tuberculin testing. However this figure falls to 24% (11-42) of recurrent breakdowns under our alternative model. Under both models the estimated extrinsic force of infection increases with the burden of missed infection. Hence, improved herd-level testing is unlikely to reduce recurrence unless this extrinsic infectious pressure is simultaneously addressed.

Bovine tuberculosis (bTB) is one of the most serious economic animal health problems affecting the cattle industry in Great Britain (GB), with incidence in cattle herds increasing since the mid-1980s. The single intradermal comparative cervical tuberculin (SICCT) test is the primary screening test in the bTB surveillance and control programme in GB and Ireland. The sensitivity (ability to detect infected cattle) of this test is central to the efficacy of the current testing regime, but most previous studies that have estimated test sensitivity (relative to the number of slaughtered cattle with visible lesions [VL] and/or positive culture results) lacked post-mortem data for SICCT test-negative cattle. The slaughter of entire herds ("whole herd slaughters" or "depopulations") that are infected by bTB are occasionally conducted in GB as a last-resort control measure to resolve intractable bTB herd breakdowns. These provide additional post-mortem data for SICCT test-negative cattle, allowing a rare opportunity to calculate the animal-level sensitivity of the test relative to the total number of SICCT test-positive and negative VL animals identified post-mortem (rSe). In this study, data were analysed from 16 whole herd slaughters (748 SICCT test-positive and 1031 SICCT test-negative cattle) conducted in GB between 1988 and 2010, using a bayesian hierarchical model. The overall rSe estimate of the SICCT test at the severe interpretation was 85% (95% credible interval [CI]: 78-91%), and at standard interpretation was 81% (95% CI: 70-89%). These estimates are more robust than those previously reported in GB due to inclusion of post-mortem data from SICCT test-negative cattle.

UNLABELLED: a case-control investigation was undertaken to determine management and health related factors associated with pleurisy in slaughter pigs in England and Wales. METHODS: the British Pig Executive Pig Health Scheme database of abattoir pathology was used to identify 121 case (>10% prevalence of pleurisy on 3 or more assessment dates in the preceding 24 months) and 121 control units (≤5% prevalence of pleurisy on 3 or more assessment dates in the preceding 24 months). Farm data were collected by postal questionnaire. Data from respondents (70 cases and 51 controls) were analysed using simple logistic regression models with Bonferroni corrections. Limited multivariate analyses were also performed to check the robustness of the overall conclusions. RESULTS AND CONCLUSIONS: Management factors associated with increased odds of pleurisy included no all-in all-out pig flow (OR 9.3, 95% confidence interval [CI]: 3.3-29), rearing of pigs with an age difference of >1 month in the same airspace (OR 6.5 [2.8-17]) and repeated mixing (OR 2.2 [1.4-3.8]) or moving (OR 2.2 [1.5-3.4]) of pigs during the rearing phase. Those associated with decreased odds of pleurisy included filling wean-to-finish or grower-to-finish systems with piglets from ≤3 sources (OR 0.18 [0.07-0.41]) compared to farrow-to-finish systems, cleaning and disinfecting of grower (ORs 0.28 [0.13-0.61] and 0.29 [0.13-0.61]) and finisher (ORs 0.24 [0.11-0.51] and 0.2 [0.09-0.44]) accommodation between groups, and extended down time of grower and finisher accommodation (OR 0.84 [0.75-0.93] and 0.86 [0.77-0.94] respectively for each additional day of downtime). This study demonstrated the value of national-level abattoir pathology data collection systems for case control analyses and generated guidance for on-farm interventions to help reduce the prevalence of pleurisy in slaughter pigs.

Bovine tuberculosis is endemic in cattle herds in Great Britain, with a substantial economic impact. A reservoir of Mycobacterium bovis within the Eurasian badger (Meles meles) population is thought to have hindered disease control. Cattle herd incidents, termed breakdowns, that are either 'prolonged' (lasting ≥ 240 days) or 'recurrent' (with another breakdown within a specified time period) may be important foci for onward spread of infection. They drain veterinary resources and can be demoralising for farmers. Randomised Badger Culling Trial (RBCT) data were re-analysed to examine the effects of two culling strategies on breakdown prolongation and recurrence, during and after culling, using a Bayesian hierarchical model. Separate effect estimates were obtained for the 'core' trial areas (where culling occurred) and the 'buffer' zones (up to 2 km outside of the core areas). For breakdowns that started during the culling period, 'reactive' (localised) culling was associated with marginally increased odds of prolongation, with an odds ratio (OR) of 1.7 (95% credible interval [CI] 1.1-2.4) within the core areas. This effect was not present after the culling ceased. There was no notable effect of 'proactive' culling on prolongation. In contrast, reactive culling had no effect on breakdown recurrence, though there was evidence of a reduced risk of recurrence in proactive core areas during the culling period (ORs and 95% CIs: 0.82 (0.64-1.0) and 0.69 (0.54-0.86) for 24- and 36-month recurrence respectively). Again these effects were not present after the culling ceased. There seemed to be no effect of culling on breakdown prolongation or recurrence in the buffer zones. These results suggest that the RBCT badger culling strategies are unlikely to reduce either the prolongation or recurrence of breakdowns in the long term, and that reactive strategies (such as employed during the RBCT) are, if anything, likely to impact detrimentally on breakdown persistence.

The development of modern and affordable sequencing technologies has allowed the study of viral populations to an unprecedented depth. This is of particular interest for the study of within-host RNA viral populations, where variation due to error-prone polymerases can lead to immune escape, antiviral resistance and adaptation to new host species. Methods to sequence RNA virus genomes include reverse transcription (RT) and polymerase chain reaction (PCR). RT-PCR is a molecular biology technique widely used to amplify DNA from an RNA template. The method itself relies on the in vitro synthesis of copy DNA from RNA followed by multiple cycles of DNA amplification. However, this method introduces artefactual errors that can act as confounding factors when the sequence data are analysed. Although there are a growing number of published studies exploring the intra- and inter-host evolutionary dynamics of RNA viruses, the complexity of the methods used to generate sequences makes it difficult to produce probabilistic statements about the likely sources of observed sequence variants. This complexity is further compounded as both the depth of sequencing and the length of the genome segment of interest increase. Here we develop a bayesian method to characterise and differentiate between likely structures for the background viral population. This approach can then be used to identify nucleotide sites that show evidence of change in the within-host viral population structure, either over time or relative to a reference sequence (e.g. an inoculum or another source of infection), or both, without having to build complex evolutionary models. Identification of these sites can help to inform the design of more focussed experiments using molecular biology tools, such as site-directed mutagenesis, to assess the function of specific amino acids. We illustrate the method by applying to datasets from experimental transmission of equine influenza, and a pre-clinical vaccine trial for HIV-1.

The interaction between Salmonella enterica and the host immune system is complex. The outcome of an infection is the result of a balance between the in vivo environment where the bacteria survive and grow and the regulation of fitness genes at a level sufficient for the bacteria to retain their characteristic rate of growth in a given host. Using bacteriological counts from tissue homogenates and fluorescence microscopy to determine the spread, localization, and distribution of S. enterica in the tissues, we show that, during a systemic infection, S. enterica adapts to the in vivo environment. The adaptation becomes a measurable phenotype when bacteria that have resided in a donor animal are introduced into a recipient naïve animal. This adaptation does not confer increased resistance to early host killing mechanisms but can be detected as an enhancement in the bacterial net growth rate later in the infection. The enhanced growth rate is lost upon a single passage in vitro, and it is therefore transient and not due to selection of mutants. The adapted bacteria on average reach higher intracellular numbers in individual infected cells and therefore have patterns of organ spread different from those of nonadapted bacteria. These experiments help in developing an understanding of the influence of passage in a host on the fitness and virulence of S. enterica.

Several classes and multiple subclasses of immunoglobulins are produced towards protein and polysaccharide antigens in response to Salmonella infection and play a key role in protection against systemic disease. The targeting of Salmonella to Fc receptors (FcR) on phagocytes is a key step in the antibody-mediated antibacterial functions of host cells. We wished to compare the relative efficiency of different human IgG subclasses, which targeted the Salmonella enterica OmpA surface protein in modulating the interaction of bacteria with human phagocytes. To this end, we developed a novel system by tagging OmpA with a foreign CD52 mimotope (TSSPSAD) and opsonizing the bacteria with a panel of humanized CD52 antibodies that share the same antigen-binding V-region, but have constant regions of different subclasses. Our data revealed that opsonization with all the IgG subclasses increases Salmonella uptake by human phagocytes. IgG3 resulted in the highest level of bacterial uptake and the highest average bacterial load per infected cell, which was closely followed by IgG1, then IgG4 and lastly IgG2. Phagocytosis mediated by IgG1, IgG3 and IgG4 had a higher dependency on FcγRI than FcγRIIA, whereas IgG2-mediated phagocytosis required FcγRIIA more than FcγRI. The results show that IgG binding to OmpA increases the uptake of Salmonella by human phagocytic cells and that the efficiency of this process depends both on the subclass of the IgG and the type of FcR that is available for antibody binding.

Salmonella enterica serovar Typhi, the agent of typhoid fever in humans, expresses the surface Vi polysaccharide antigen that contributes to virulence. However, Vi expression can also be detrimental to some key steps of S. Typhi infectivity, for example, invasion, and Vi is the target of protective immune responses. We used a strain of S. Typhimurium carrying the whole Salmonella pathogenicity island 7 (SPI-7) to monitor in vivo Vi expression within phagocytic cells of mice at different times after systemic infection. We also tested whether it is possible to modulate Vi expression via the use of in vivo-inducible promoters and whether this would trigger anti-Vi antibodies through the use of Vi-expressing live bacteria. Our results show that Vi expression in the liver and spleen is downregulated with the progression of infection and that the Vi-negative population of bacteria becomes prevalent by day 4 postinfection. Furthermore, we showed that replacing the natural tviA promoter with the promoter of the SPI-2 gene ssaG resulted in sustained Vi expression in the tissues. Intravenous or oral infection of mice with a strain of S. Typhimurium expressing Vi under the control of the ssaG promoter triggered detectable levels of all IgG subclasses specific for Vi. Our work highlights that Vi is downregulated in vivo and provides proof of principle that it is possible to generate a live attenuated vaccine that induces Vi-specific antibodies after single oral administration.

Since the demonstration that almost 80% of human immunodeficiency virus type 1 (HIV-1) infections result from the transmission of a single variant from the donor, biological features similar to those of HIV mucosal transmission have been reported for macaques inoculated with simian immunodeficiency virus (SIV). Here we describe the early diversification events and the impact of challenge doses on viral kinetics and on the number of variants transmitted in macaques infected with the chimeric simian/human immunodeficiency virus SHIV(sf162p4). We show that there is a correlation between the dose administered and the number of variants transmitted and that certain inoculum variants are preferentially transmitted. This could provide insight into the viral determinants of transmission and could aid in vaccine development. Challenge through the mucosal route with high doses results in the transmission of multiple variants in all the animals. Such an unrealistic scenario could underestimate potential intervention measures. We thus propose the use of molecular evolution analysis to aid in the determination of challenge doses that better mimic the transmission dynamics seen in natural HIV-1 infection.

Bovine tuberculosis (bTB) is an important economic disease worldwide with implications for both animal and human health. In Great Britain the number of herds that test positive for bTB, termed "breakdowns", has increased over the last two decades. Despite more intensive testing during a breakdown, around 23% of breakdowns recur within 12 months of the previous breakdown ending, and around 38% within 24 months. These "recurrent" breakdowns may be important for onward transmission of infection. Detailed case-control data were analysed to identify factors associated with recurrence within 12 months. The model predicted 83% of all recurrent breakdowns, with a positive predictive value (PPV) of 44%. A further model, restricted to data currently available nationally, was not sufficient to predict recurrence reliably; at a sensitivity of 72-76%, the PPV was 33-36%, when validated on independent data. Factors identified to be associated with recurrence are consistent with previous studies; namely, the number of reactors, a recent bTB history in the herd and a lack of association with the confirmation status of the initial breakdown. These variables are indicative of a higher level of infection or residual infection, and could be useful in the future development of predictive models for bTB recurrence.

Infection of chickens with avian influenza virus poses a global threat to both poultry production and human health that is not adequately controlled by vaccination or by biosecurity measures. A novel alternative strategy is to develop chickens that are genetically resistant to infection. We generated transgenic chickens expressing a short-hairpin RNA designed to function as a decoy that inhibits and blocks influenza virus polymerase and hence interferes with virus propagation. Susceptibility to primary challenge with highly pathogenic avian influenza virus and onward transmission dynamics were determined. Although the transgenic birds succumbed to the initial experimental challenge, onward transmission to both transgenic and nontransgenic birds was prevented.

OBJECTIVE: to (1) discover kinematic variables that differ between dogs with cranial cruciate ligament rupture (CCLR) and control dogs and (2) to identify suitable outcome variables for longitudinal studies on the effects of surgical interventions for CCLR in dogs. STUDY DESIGN: in vivo case-control comparison. ANIMALS: Dogs with unilateral CCLR (n=16), and no other detectable neurologic or orthopedic disease and 10 dogs without CCLR, neurologic, or orthopedic disease. METHODS: Kinematic data was collected from dogs as they walked at constant speed on a treadmill, using an infrared camera system and adhesive reflective markers applied to the pelvic limbs. Data on 5 selected variables was extracted and comparisons made between paired pelvic limbs in both CCLR and control dogs and between control and CCLR dogs. RESULTS: Comparisons in CCLR dogs revealed significant differences between affected and unaffected limbs for many examined variables; the largest differences were in paw velocity and stifle angular velocity. There were highly significant differences between CCLR and control dogs when examining paired limb ratios for 4 of the variables, the largest differences were in stride length and paw velocity. CONCLUSIONS: Kinematic analysis provides straightforward and objective methods for defining the lameness associated with CCLR in dogs. Paw velocity and stride length were most notably reliable and not susceptible to systematic alterations in stifle joint dynamics that might be associated with specific surgical procedures. CLINICAL RELEVANCE: This study identifies several variables that can provide the objective measurements essential to evaluate the efficacy of surgical interventions for CCLR in dogs.

Objective-To compare survival of dogs with a congenital portosystemic shunt (CPSS) that received medical or surgical treatment. Design-Prospective cohort study. Animals-126 client-owned dogs with a single CPSS. Procedures-Dogs were examined at 1 of 3 referral clinics, and a single CPSS was diagnosed in each. Dogs received medical or surgical treatment without regard to signalment, clinical signs, or results of hematologic or biochemical analysis. Survival data were analyzed via a Cox regression model. Results-During a median follow-up period of 579 days.18 of 126 dogs died as a result of CPSS. Dogs treated via surgical intervention survived significantly longer than did those treated medically. Hazard ratio for medical versus surgical treatment of CPSS (for the treatment-only model) was 2.9 (95% confidence interval, 1.1 to 7.2). Age at CPSS diagnosis did not affect survival. Conclusions and Clinical Relevance-Both medical and surgical treatment can be used to achieve long-term survival of dogs with CPSS, although results of statistical analysis supported the widely held belief that surgery is preferable to medical treatment. However, the study population consisted of dogs at referral clinics, which suggested that efficacy of medical treatment may have been underestimated. Although surgical intervention was associated with a better chance of long-term survival, medical management provided an acceptable first-line option. Age at examination did not affect survival, which implied that early surgical intervention was not essential. Dogs with CPSS that do not achieve acceptable resolution with medical treatment can subsequently be treated surgically.

Determining the evolutionary basis of cross-species transmission and immune evasion is key to understanding the mechanisms that control the emergence of either new viruses or novel antigenic variants with pandemic potential. The hemagglutinin glycoprotein of influenza a viruses is a critical host range determinant and a major target of neutralizing antibodies. Equine influenza virus (EIV) is a significant pathogen of the horse that causes periodical outbreaks of disease even in populations with high vaccination coverage. EIV has also jumped the species barrier and emerged as a novel respiratory pathogen in dogs, canine influenza virus. We studied the dynamics of equine influenza virus evolution in horses at the intrahost level and how this evolutionary process is affected by interhost transmission in a natural setting. To this end, we performed clonal sequencing of the hemagglutinin 1 gene derived from individual animals at different times postinfection. Our results show that despite the population consensus sequence remaining invariant, genetically distinct subpopulations persist during the course of infection and are also transmitted, with some variants likely to change antigenicity. We also detected a natural case of mixed infection in an animal infected during an outbreak of equine influenza, raising the possibility of reassortment between different strains of virus. In sum, our data suggest that transmission bottlenecks may not be as narrow as originally perceived and that the genetic diversity required to adapt to new host species may be partially present in the donor host and potentially transmitted to the recipient host.

Bovine tuberculosis (bTB) is an important notifiable disease in cattle in Great Britain (GB), and is subject to statutory control measures. Despite this, disease incidence has increased since the mid-1980s, and around 30% of herd breakdowns continue for more than 240 days. This is twice the shortest possible time for confirmed breakdowns to test clear from infection (≈120 days), and four times the shortest possible time for unconfirmed breakdowns (≈60 days). These "prolonged" breakdowns consume substantial resources and may act as an ongoing source of infection. It is not clear why some breakdowns become prolonged. Existing detailed case-control data have been re-analysed to determine risk factors for breakdowns lasting longer than 240 days, the strongest of which was the confirmation status of the breakdown: OR 12.6 (95%CI: 6.7-25.4). A further model restricted to data available early on in a breakdown for all breakdowns nationally, can predict 82-84% of prolonged breakdowns with a positive predictive value of 44-49% when validated using existing national datasets over a 4-year period. Identification of prolonged breakdowns at an earlier stage could help to target bTB controls in GB.

Bacteria of the species Salmonella enterica cause a range of life-threatening diseases in humans and animals worldwide. The within-host quantitative, spatial, and temporal dynamics of S. enterica interactions are key to understanding how immunity acts on these infections and how bacteria evade immune surveillance. In this study, we test hypotheses generated from mathematical models of in vivo dynamics of Salmonella infections with experimental observation of bacteria at the single-cell level in infected mouse organs to improve our understanding of the dynamic interactions between host and bacterial mechanisms that determine net growth rates of S. enterica within the host. We show that both bacterial and host factors determine the numerical distributions of bacteria within host cells and thus the level of dispersiveness of the infection.

A cohort study of 174 flat-coated retrievers was undertaken to establish the importance of cancer in flat coat mortality in terms of the prevalence of neoplasia in the breed and also the relative effect of cancer on lifespan in relation to other forms of mortality. Dogs aged 2-7 years were recruited in 1996 and followed until 2007. An annual health census was used to collect the data. Two dogs were lost to follow-up and 72 dogs (42%) died from confirmed neoplasia. Twenty dogs (11.6%) died of unconfirmed tumours and 61 (35%) died from non-neoplastic conditions. The cause of death was unknown for 19 dogs. Soft tissue sarcoma (especially histiocytic sarcoma) was the predominant cancer type, affecting 32 dogs (44% of neoplasms). Six dogs died with malignant melanoma and three with lymphoma. Median age at death was 9 years for dogs with tumours (eight for sarcoma patients) and 12 years for non-neoplastic fatalities. The results confirm that soft tissue sarcoma, particularly histiocytic sarcoma, is a major cause of mortality in this breed.

LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57-66 and 82-89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure (Cell 130: 1071-1082) of the ligand-bound TLR1/2 ectodomain heterodimer.

Mechanistic determinants of bacterial growth, death, and spread within mammalian hosts cannot be fully resolved studying a single bacterial population. They are also currently poorly understood. Here, we report on the application of sophisticated experimental approaches to map spatiotemporal population dynamics of bacteria during an infection. We analyzed heterogeneous traits of simultaneous infections with tagged Salmonella enterica populations (wild-type isogenic tagged strains [WITS]) in wild-type and gene-targeted mice. WITS are phenotypically identical but can be distinguished and enumerated by quantitative PCR, making it possible, using probabilistic models, to estimate bacterial death rate based on the disappearance of strains through time. This multidisciplinary approach allowed us to establish the timing, relative occurrence, and immune control of key infection parameters in a true host-pathogen combination. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs, a process controlled by host antimicrobial mechanisms. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs via bacteraemia and further stochastic selection. This approach provides us with an unprecedented outlook on the pathogenesis of S. enterica infections, illustrating the complex spatial and stochastic effects that drive an infectious disease. The application of the novel method that we present in appropriate and diverse host-pathogen combinations, together with modelling of the data that result, will facilitate a comprehensive view of the spatial and stochastic nature of within-host dynamics. © 2008 Grant et al.

Mechanistic determinants of bacterial growth, death, and spread within mammalian hosts cannot be fully resolved studying a single bacterial population. They are also currently poorly understood. Here, we report on the application of sophisticated experimental approaches to map spatiotemporal population dynamics of bacteria during an infection. We analyzed heterogeneous traits of simultaneous infections with tagged Salmonella enterica populations (wild-type isogenic tagged strains [WITS]) in wild-type and gene-targeted mice. WITS are phenotypically identical but can be distinguished and enumerated by quantitative PCR, making it possible, using probabilistic models, to estimate bacterial death rate based on the disappearance of strains through time. This multidisciplinary approach allowed us to establish the timing, relative occurrence, and immune control of key infection parameters in a true host-pathogen combination. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs, a process controlled by host antimicrobial mechanisms. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs via bacteraemia and further stochastic selection. This approach provides us with an unprecedented outlook on the pathogenesis of S. enterica infections, illustrating the complex spatial and stochastic effects that drive an infectious disease. The application of the novel method that we present in appropriate and diverse host-pathogen combinations, together with modelling of the data that result, will facilitate a comprehensive view of the spatial and stochastic nature of within-host dynamics.

During systemic disease in mice, Salmonella enterica grows intracellularly within discrete foci of infection in the spleen and liver. In concomitant infections, foci containing different S. enterica strains are spatially separated. We have investigated whether functional interactions between bacterial populations within the same host can occur despite the known spatial separation of the foci and independence of growth of salmonellae residing in different foci. In this study we have demonstrated that bacterial numbers of virulent S. enterica serovar Typhimurium C5 strain in mouse tissues can be increased by the presence of the attenuated aroA S. Typhimurium SL3261 vaccine strain in the same tissue. Disease exacerbation does not require simultaneous coinjection of the attenuated bacteria. SL3261 can be administered up to 48 hr after or 24 hr before the administration of C5 and still determine higher tissue numbers of the virulent bacteria. This indicates that intravenous administration of a S. enterica vaccine strain could potentially exacerbate an established infection with wild-type bacteria. These data also suggest that the severity of an infection with a virulent S. enterica strain can be increased by the prior administration of a live attenuated vaccine strain if infection occurs within 48 hr of vaccination. Exacerbation of the growth of C5 requires Toll-like receptor 4-dependent interleukin-10 production with the involvement of both Toll/interleukin-1 receptor-domain-containing adaptor inducing interferon-beta and myeloid differentiation factor 88.

A symposium discussing collaborative research work on infectious diseases dynamics was held at Queens' College, University of Cambridge on 25 October 2006.