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Background Identifying the mechanisms driving disease risk is challenging for multi-host pathogens, such as Borrelia burgdorferi sensu lato (s.l.), the tick-borne bacteria causing Lyme disease. Deer are tick reproduction hosts but do not transmit B. burgdorferi s.l., whereas rodents and birds are competent transmission hosts. Here, we use a long-term deer exclosure experiment to test three mechanisms for how high deer density might shape B. burgdorferi s.l. prevalence in ticks: increased prevalence due to higher larval tick densities facilitating high transmission on rodents (M1); alternatively, reduced B. burgdorferi s.l. prevalence because more larval ticks feed on deer rather than transmission-competent rodents (dilution effect) (M2), potentially due to ecological cascades, whereby higher deer grazing pressure shortens vegetation which decreases rodent abundance thus reducing transmission (M3). Methods In a large enclosure where red deer stags were kept at high density (35.5 deer km −2 ), we used an experimental design consisting of eight plots of 0.23 ha, four of which were fenced to simulate the absence of deer and four that were accessible to deer. In each plot we measured the density of questing nymphs and nymphal infection prevalence in spring, summer and autumn, and quantified vegetation height and density, and small mammal abundance. Results Prevalence tended to be lower, though not conclusively so, in high deer density plots compared to exclosures (predicted prevalence of 1.0% vs 2.2%), suggesting that the dilution and cascade mechanisms might outweigh the increased opportunities for transmission mechanism. Presence of deer at high density led to shorter vegetation and fewer rodents, consistent with an ecological cascade. However, Lyme disease hazard (density of infected I. ricinus nymphs) was five times higher in high deer density plots due to tick density being 18 times higher. Conclusions High densities of tick reproduction hosts such as deer can drive up vector-borne disease hazard, despite the potential to simultaneously reduce pathogen prevalence. This has implications for environmental pathogen management and for deer management, although the impact of intermediate deer densities now needs testing. Graphical abstract

Ticks are the main arthropod vector of pathogens to humans and livestock in the British Isles. Despite their role as a vector of disease, many aspects of tick biology, ecology, and microbial association are poorly understood. To address this, we investigated the composition of the microbiome of adult and nymphal Ixodes ricinus ticks. The ticks were collected on a dairy farm in Southwest England and RNA extracted for whole genome sequencing. Sequences were detected from a range of microorganisms, particularly tick-associated viruses, bacteria, and nematodes. A majority of the viruses were attributed to phlebo-like and nairo-like virus groups, demonstrating a high degree of homology with the sequences present in I. ricinus from mainland Europe. A virus sharing a high sequence identity with Chimay rhabdovirus, previously identified in ticks from Belgium, was detected. Further investigations of I. ricinus ticks collected from additional sites in England and Wales also identified Chimay rhabdovirus viral RNA with varying prevalence in all tick populations. This suggests that Chimay rhabdovirus has a wide distribution and highlights the need for an extended exploration of the tick microbiome in the United Kingdom (UK).

Background With medically important arboviruses such as West Nile virus (WNV) circulating in Europe and Usutu virus (USUV) currently present in the UK, it is imperative to identify areas in the UK at risk of establishment and spread of these viruses. Here, we describe a comprehensive nationwide field surveillance study conducted during July 2023 to map the distribution of the WNV and USUV competent vectors: Culex pipiens biotype pipiens , Culex pipiens biotype molestus and Culex torrentium , across England and Wales. Methods Mosquitoes were sampled for 3 trap nights (3TN) at 200 sites in rural, urban and suburban settings, selected using a lattice plus close pairs surveillance design. Field-caught samples were analysed using morphological and molecular approaches. Results A total of 2157 adult mosquitoes of the Culex genus were collected. Culex pipiens biotype pipiens dominated the resident Culex populations, comprising 1478 (95.8%) out of 1543 mosquitoes with confirmed molecular species identity. Culex torrentium were present in much lower numbers, with only 38 (2.5%) identified mostly and in the central and southwestern regions. Only five of the biotype molestus (0.3%) were identified in this study; however these were found in localities outside of London and in a rural location, with the biotype previously having been associated with human-made habitats. This study also found that hybrids of the pipiens and molestus biotypes are more widespread than previously recorded. In total, 22 hybrids (1.4%) were identified from counties as far west as Cornwall and as far north as Suffolk. Conclusions Coupled with reviews of previous UK Culex sp. surveys, this study provides essential data for the vectorial component of risk modelling of WNV and USUV in the UK, furthering the country’s preparedness for incursions of vector-borne diseases in the future. Graphical Abstract

Anaplasma phagocytophilum (A. phagocytophilum) is the aetiological agent of tick-borne fever in cattle and sheep, and granulocytic anaplasmosis in human and dogs. Livestock, companion animal and human infections with A. phagocytophilum have been reported globally. Across England and Wales, two isolates (called ecotypes) have been reported in ticks. This study examined A. phagocytophilum isolates present in livestock and wildlife in Great Britain (GB), with a particular focus on cattle. Clinical submissions (EDTA blood) from cattle (n = 21) and sheep (n = 3) were received by APHA for tick-borne disease testing and the animals were confirmed to be infected with A. phagocytophilum using a PCR targeting the Msp2 gene. Further submissions from roe deer (n = 2), red deer (n = 2) and Ixodes ricinus ticks (n = 22) were also shown to be infected with A. phagocytophilum. Subsequent analysis using a nested PCR targeting the groEL gene and sequencing confirmed the presence of ecotype I in cattle, sheep, red deer and Ixodes ricinus, and ecotype II in roe deer and I. ricinus removed from deer carcasses. Despite the presence of two ecotypes, widely distributed in ticks from England and Wales, only ecotype I was detected in cattle in this study.

The largest land use change in Europe is woodland expansion, through planting and natural regeneration. Unforeseen consequences of this could include changes in environmental hazards, such as exposure to parasites and pathogens. Tick‐borne Lyme disease is the most prevalent tick‐borne disease in the northern hemisphere and is often associated with woodlands. Therefore, to inform the planning and management of expanding woodlands, we test how land covers that reflect different types and stages of the woodland expansion process, along with their deer management, impact tick densities and Lyme disease hazard (density of infected nymphs). We also test whether differences in rodent abundance play a mechanistic role in explaining differences in Lyme disease hazard. In Northwest Scotland, a touristic area undergoing woodland expansion, we recorded deer management, rodent densities, Ixodes ricinus nymph densities, pathogen prevalence and Lyme disease hazard between open moorland, young pine and mature pine, and birch and spruce. These represent pre‐, early and late stage woodland establishments, and the three woodland types in the region. Rodents, ticks, pathogen prevalence and Lyme disease hazard were generally lowest in moorland and young pine and highest in mature woodland, especially birch, although variability was high. Deer management reduced tick densities and, marginally, Lyme disease hazard. There was insufficient evidence for rodents increasing Lyme disease hazard, but rodents augmented tick densities and the most abundant Lyme disease pathogen was that transmitted by rodents. Practical implication. woodland expansion could, once mature, eventually lead to higher tick densities, pathogen prevalence and Lyme disease hazard. Importantly, an environmental solution could be to control deer populations. Woodland expansion may eventually, once the trees mature, increase the risk of ticks and Lyme disease. However, controlling deer populations could be a solution to mitigate this issue.

To better understand vector‐borne disease dynamics, knowledge of the ecological interactions between animal hosts, vectors, and pathogens is needed. The effects of hosts on disease hazard depends on their role in driving vector abundance and their ability to transmit pathogens. Theoretically, a host that cannot transmit a pathogen could dilute pathogen prevalence but increase disease hazard if it increases vector population size. In the case of Lyme disease, caused by Borrelia burgdorferi s.l. and vectored by Ixodid ticks, deer may have dual opposing effects on vectors and pathogen: deer drive tick population densities but do not transmit B. burgdorferi s.l. and could thus decrease or increase disease hazard. We aimed to test for the role of deer in shaping Lyme disease hazard by using a wide range of deer densities while taking transmission host abundance into account. We predicted that deer increase nymphal tick abundance while reducing pathogen prevalence. The resulting impact of deer on disease hazard will depend on the relative strengths of these opposing effects. We conducted a cross‐sectional survey across 24 woodlands in Scotland between 2017 and 2019, estimating host (deer, rodents) abundance, questing Ixodes ricinus nymph density, and B. burgdorferi s.l. prevalence at each site. As predicted, deer density was positively associated with nymph density and negatively with nymphal infection prevalence. Overall, these two opposite effects canceled each other out: Lyme disease hazard did not vary with increasing deer density. This demonstrates that, across a wide range of deer and rodent densities, the role of deer in amplifying tick densities cancels their effect of reducing pathogen prevalence. We demonstrate how noncompetent host density has little effect on disease hazard even though they reduce pathogen prevalence, because of their role in increasing vector populations. These results have implications for informing disease mitigation strategies, especially through host management. Conceptual diagram illustrating the predicted effects of deer and rodent densities on the density of nymphs, nymphal infection prevalence and Lyme disease hazard.

Ticks are the main arthropod vector of pathogens to humans and livestock in the British Isles. Despite their role as a vector of disease, many aspects of tick biology, ecology, and microbial association are poorly understood. To address this, we investigated the composition of the microbiome of adult and nymphal Ixodes ricinus ticks. The ticks were collected on a dairy farm in Southwest England and RNA extracted for whole genome sequencing. Sequences were detected from a range of microorganisms, particularly tick-associated viruses, bacteria, and nematodes. A majority of the viruses were attributed to phlebo-like and nairo-like virus groups, demonstrating a high degree of homology with the sequences present in I. ricinus from mainland Europe. A virus sharing a high sequence identity with Chimay rhabdovirus, previously identified in ticks from Belgium, was detected. Further investigations of I. ricinus ticks collected from additional sites in England and Wales also identified Chimay rhabdovirus viral RNA with varying prevalence in all tick populations. This suggests that Chimay rhabdovirus has a wide distribution and highlights the need for an extended exploration of the tick microbiome in the United Kingdom (UK).

Anaplasma phagocytophilum (A. phagocytophilum) is the aetiological agent of tick-borne fever in cattle and sheep, and granulocytic anaplasmosis in human and dogs. Livestock, companion animal and human infections with A. phagocytophilum have been reported globally. Across England and Wales, two isolates (called ecotypes) have been reported in ticks. This study examined A. phagocytophilum isolates present in livestock and wildlife in Great Britain (GB), with a particular focus on cattle. Clinical submissions (EDTA blood) from cattle (n = 21) and sheep (n = 3) were received by APHA for tick-borne disease testing and the animals were confirmed to be infected with A. phagocytophilum using a PCR targeting the Msp2 gene. Further submissions from roe deer (n = 2), red deer (n = 2) and Ixodes ricinus ticks (n = 22) were also shown to be infected with A. phagocytophilum. Subsequent analysis using a nested PCR targeting the groEL gene and sequencing confirmed the presence of ecotype I in cattle, sheep, red deer and Ixodes ricinus, and ecotype II in roe deer and I. ricinus removed from deer carcasses. Despite the presence of two ecotypes, widely distributed in ticks from England and Wales, only ecotype I was detected in cattle in this study.

Assessing the risk of tick-borne disease in areas with high visitor numbers is important from a public health perspective. Evidence suggests that tick presence, density, infection prevalence and the density of infected ticks can vary between habitats within urban green space, suggesting that the risk of Lyme borreliosis transmission can also vary. This study assessed nymph density, Borrelia prevalence and the density of infected nymphs across a range of habitat types in nine parks in London which receive millions of visitors each year. Ixodes ricinus were found in only two of the nine locations sampled, and here they were found in all types of habitat surveyed. Established I. ricinus populations were identified in the two largest parks, both of which had resident free-roaming deer populations. Highest densities of nymphs (15.68 per 100 m2) and infected nymphs (1.22 per 100 m2) were associated with woodland and under canopy habitats in Richmond Park, but ticks infected with Borrelia were found across all habitat types surveyed. Nymphs infected with Borrelia (7.9%) were only reported from Richmond Park, where Borrelia burgdorferi sensu stricto and Borrelia afzelii were identified as the dominant genospecies. Areas with short grass appeared to be less suitable for ticks and maintaining short grass in high footfall areas could be a good strategy for reducing the risk of Lyme borreliosis transmission to humans in such settings. In areas where this would create conflict with existing practices which aim to improve and/or meet historic landscape, biodiversity and public access goals, promoting public health awareness of tick-borne disease risks could also be utilised.

Emergence of zoonoses are driven by multiple factors, ranging from climate change to urbanization and human behaviours. Because many zoonotic pathogens are maintained in wild reservoir hosts, these factors of emergence may affect disease risk by changing host community parameters. Thus, it is important to understand the effect of host community composition on disease risk. This is particularly relevant for vector-borne zoonoses as host community composition might affect both reservoir host and vector populations. In the northern hemisphere, Lyme disease, a zoonosis caused by the bacterial complex Borrelia burgdorferi sensu lato, is the most prevalent vector-borne disease affecting humans. Transmitted by Ixodid ticks, its epidemiological cycle is complex and depends on environmental factors and host community composition, which together, influence both tick survival and the prevalence of B. burgdorferi s.l. Small mammals are competent reservoir hosts for B. afzelii, a genospecies belonging to the B. burgdorferi s.l. complex, while birds can transmit B. valaisiana and B. garinii, two other genospecies in the complex. Thus, pathogen prevalence will depend on the abundance of competent reservoir hosts in an environment. Deer species, on the other hand, are non-competent hosts and could, theoretically, lower B. burgdorferi s.l. prevalence by diverting immature ticks from feeding on competent reservoir hosts, a concept called the dilution effect. However, deer also act as the main tick reproduction hosts, feeding adult female ticks, and can therefore maintain high tick populations. In this thesis, I used a range of approaches (large-scale cross-sectional survey, deer exclosure and wood mouse supplementary feeding experiment) to test hypotheses and understand how host community composition drives Lyme disease hazard (defined here as the density of infected nymphal ticks) in Scotland and to investigate the drivers of host communities. I investigated how the ratio of small mammals to deer affected Lyme disease hazard by conducting a large cross-sectional survey across sites selected to specifically cover the full range of deer densities (Chapter 2) and collecting data on host abundance (deer and small mammals), tick density and pathogen prevalence. There was a positive association between the density of infected nymphs (DIN) for B. afzelii and deer density, regardless of small mammal abundance. I also observed a positive association between deer density and quest in deer density was also negatively associated with human activity, which therefore has the potential to impact Lyme disease hazard. Having shown how the full range of deer densities shapes Lyme disease hazard, which also suggested that deer may affect small mammal densities (Chapter 2), I then wanted to investigate further the possibility that high deer densities may affect Lyme disease by causing ecological cascades, through an impact on vegetation and small mammals (Chapter 3). I used an experimental design consisting of replicated fenced deer exclosures to investigate the effects of high deer density versus deer absence on Lyme disease hazard through ecological cascades. Consistent with my predictions, high deer density plots had 18 times more questing nymphs compared to plots where deer were absent. High deer density plots also had 13 times fewer small mammals and were associated with shorter and sparser vegetation and shorter trees, highlighting the impacts of browsing pressure by deer on small mammal communities. I found that the reduction in competent host abundance had repercussions on B. burgdorferi s.l. prevalence in questing ticks, which was twice as high in deer exclusion plots (2.2% in deer exclusion plots vs 1.0% in high deer density plots). Despite the negative effect of high deer density on B. burgdorferi s.l. prevalence, DIN was five times higher in high deer density plots. These results demonstrate that the positive effect of deer on tick density can outweigh their negative effect on B. burgdorferi s.l. prevalence caused by a dilution effect and through their negative effects on small mammals. In chapters 2 and 3, I found that deer appeared to be more important than small mammals in driving DIN, through their strong role as tick reproduction hosts, irrespective of any dilution effects on prevalence. However, few of my sites had high small mammal densities while many had high deer densities. I therefore wanted to test the effect of a variation in small mammal abundance on ticks, B. burgdorferi s.l. prevalence and DIN using a supplementary feeding experiment to increase small mammal densities. More specifically, the objective for this study (Chapter 4) was to understand the effects of a change in food resources on small mammal populations and the repercussions on Lyme disease hazard. Fluctuations in resources availability are common occurrences in nature so it is important to understand the consequences this could have for disease hazard. This involved an experimental design where two trapping grids out of four were supplemented with food for two consecutive years. Density of live-trapped wood mice (Apodemus sylvaticus) was twice as high in food supplemented grids during the year of treatment. Food supplementation per se, which reflects what would happen after a mast seeding event, did not affect the density of nymphs, B. burgdorferi s.l. prevalence or DIN. However, there was a positive correlation between wood mouse abundance and questing nymph abundance the following spring. I hypothesised a negative association between wood mouse abundance and the prevalence of the bird associated B. garinii and B. valaisiana (dilution effect where more ticks would be feeding on mice instead of birds) but I did not find evidence for that. Unexpectedly, there was a positive association between wood mouse abundance and DIN for B. garinii and B.valaisiana the following spring. This could occur either if mice acted like deer did in Chapters 2 and 3, as a tick amplification host, since they increased nymph density but not B. garinii and B. valaisiana prevalence and/or if the food supplementation that increased mice also increased birds, which I did not measure. Even though food supplementation per se did not have a direct impact on DIN, wood mouse abundance had a positive effect on tick density and DIN for B. garinii and B. valaisiana and these results highlight how variations in food resources can affect small mammals and Lyme disease hazard. The cross-sectional study (Chapter 2) is the first, to my knowledge, to test how the ratio of competent reservoirs hosts (small mammals) to non-competent hosts (deer) affects Lyme disease hazard, whilst also specifically selecting sites with the full range of deer densities to robustly test the dilution effect. While I predicted that DIN should be modulated by variations in the density of both hosts, my results really pointed to deer density being the key component driving Lyme disease hazard. This result, which was further confirmed in Chapter 3, could be used for deer management decisions in Scottish woodlands. This thesis also demonstrates the complexity of Lyme disease ecology: not only due to the multiple host types and multiple pathogens in the burgdorferi s.l. complex, but also in terms of how the hosts themselves interact and affect each other’s distributions and densities. There is now clearly a need for further research to better understand the mechanisms driving Lyme disease hazard, including interspecific interactions and other drivers suggested in this thesis, such as human disturbance and food resources, that affect host abundance. Combining experimental designs and large-scale surveys allowed a better understanding of the effects of deer and small mammals simultaneously on Lyme disease. Indeed, the experimental designs (Chapters 3 and 4) offered the chance to vary the density of one host species and observe the cascading effects on other hosts, tick density and burgdorferi s.l. prevalence. Results from the cross sectional-survey (Chapter 2) not only corroborated the key associations observed but, importantly, allowed me to robustly test the dilution effect by examining the effects of a wide range of deer densities in the natural environment on DIN. The results obtained can be used to understand and predict how environmental changes (e.g. increase of resources, increase of human activity) might impact host species and Lyme disease hazard. Furthermore, they could be used to predict the effects that a change in deer management might have on tick density and DIN.