I describe procedures for the introduction of Borrelia burgdorferi, the spirochetal agent of Lyme disease, into larval and nymphal stage of the tick vector, Ixodes ricinus. The goal of this Mgr. Thesis is to find an optima system, that would reliably and reproducibly allow the infection of large numbers of ticks with the Lyme disease spirochete.

Ticks are blood feeding parasites that secrete a number of immunomodulatory factors to evade host immune response. The aim of this study was to prepare a tick salivary protein with anti-chemokine activity and to observe the influence of salivary gland extrakt on neutrophile´s chemotaxis.

Ixodes ricinus is the main vector of Lyme borreliosis and Tick-borne encephalitis in Europe. Repellents provide a practical means of protection against tick bites and can therefore reduce transmission of tick-borne diseases. In laboratory tests, pieces of cloth treated with MyggA Natural® (a commercial insect repellent) or with the essential oils of Corymbia citriodora (30%), Lavandula angustifolia (1 and 30%), Pelargonium graveolens (1 and 30%), Hyptis suaveolens (10%), Salvadora persica, Pistacia atlantica, Juniperus phoenicea (20%) and methyl jasmonate (MJ) (0.5%, 1.0%, 2.0% and 5.0%) showed strong repellent activity against I. ricinus nymphs. In a tick-infested woodland in east-central Sweden, we tested by randomized, standardised methodology the potential anti-tick repellent activity of MyggA Natural® (roll-on), two concentrations of MyggA Natural® spray, RB86 (a commercially available insect repellent for horses), the essential oil of C. citriodora and three concentrations of MJ. Each substance was dissolved in acetone and applied separately to 1 m2 cloths which were then pulled over vegetation. Nymphal tick numbers on the cloths were recorded at 10-m intervals and differed significantly between treated cloths and the untreated control and also between collectors. Volatile compounds from fresh and dried leaves of H. suaveolens and the essential oils of H. suaveolens (from Laos and Guinea Bissau) and S. persica, P. atlantica and J. phoenicea (from Libya) were collected by solid phase micro-extraction (SPME) and the constituents were identified by gas chromatography - mass spectrometry (GC-MS). Two main sesquiterpene hydrocarbons in the H. suaveolens oil were β-caryophyllene and humulene. These were modified by oxidation and sulphidation to obtain effective tick repellent compounds with lower volatility. In all three oils from Libyan plants the main monoterpene hydrocarbons were α-pinene, sabinene, β-pinene, myrcene, α-phellandrene, 4-carene, β-phellandrene and γ-terpinene. The selected plant species contained numerous volatiles known to have insecticidal, acaricidal, and/or insect repellent properties.

This study is focused on characterization of fibrinogen-related proteins (FREPs) from the tick Ixodes ricinus using molecular methods - PCR, cloning, qRT-PCR, RNA interference via dsRNA synthesis and injection, and also pathogen (Borrelia sp.) transmission on animal model.

ML (MD-2-related lipid-recognition) domain containing proteins are recognized as immune-related molecules. They do not belong among well-studied proteins in ticks although their occurence is quite often. Generally, ML proteins are involved in innate immunity processes, lipid binding and transport. Usually, expression of tick ML domain containing proteins is induced by blood feeding. Two members of the ML protein family, ML-domain containing protein and Der-p2 allergen-like protein were isolated from Ixodes ricinus and characterized for the first time.

Lyme disease is the most common vector-borne infection in the Western world with an annual incidence usually in excess of 100 cases per 100 000 people in temperate areas of the United States and Europe. Same as other infectious diseases, Lyme borreliosis wreaks havoc on the host they have invaded. B. burgdorferi, the causative agent of this disease, circulates among wildlife vertebrate hosts and Ixodes tick vectors but may sometimes infect humans. Its natural enzootic cycle usually occurs as follows: The larval/nymphal stage tick feeds on an infected host. During this engorgement, the spirochetes reach the tick gut and stay confined to it. After the tick molts into the next developmental stage, it finds a second host. The new bloodmeal triggers the spirochetes to multiply within the gut and traverse the gut endothelium in a highly organized manner. They finally disseminate through the hemocoel up to the tick salivary glands and into the new host. We studied whether B. burgdorferi is capable of reaching the tick salivary glands during the first infective feeding period in uninfected ticks.

Light microscopy was used to characterize digestive cells in the gut of the hard tick Ixodes ricinus during feeding and different phases of digestion. Indirect immunofluorescent microscopy was used to localize three digestive peptidases - cathepsin B, cathepsin L, cathepsin D in the gut of the hard tick Ixodes ricinus.

The recombinant protein c90 was prepared and polyclonal antibodies against this protein were raised. The dsRNA was made for the experiments with RNA interference. The samples from dissected tissues of dsRNA silenced ticks were tested by RT-PCR and Western blot. Results suggest that protein c90 plays a role in the tick body during the reaction to injury. Finally, another experiment with injection of water, G+ and G- bacteria into the ticks was realized. It was found that the members of the 18,7 kDa protein family can create multimers. The overexpression of silenced genes was observed during RNAi experiments despite of expected inhibition of c90 production. These results together with the bioinformatics analysis could mean that these proteins are important the physiology of tick probably as a reaction to injury. However c90 protein is produced only in the first phase of feeding which could mean that it has some role in the tick-host interaction as well.

Ticks feed on their vertebrate hosts for number of days, providing enough time for development of an effective immune response. To overcome this, tick saliva contains a complex mixture of active substances which mediate host defense mechanisms. Extremely important role at the tick-host interface is played by the protease inhibitors. In this work, we have focused on the protein named Ixocludin 2, a member of the Kunitz type/Bovine trypsin inhibitors of serine proteases, related also to the potassium channel blockers toxins. Three different expression systems (Escherichia coli, Chinese hamster ovary and Pichia pastoris) were tested to prepare an active recombinant Ixocludin 2, out of which, only bacterial system was in part successful.

La tique Ixodes ricinus est un vecteur de maladies chez l’animal et chez l’Homme d’importance majeure en Europe. Le réchauffement climatique modifiera probablement son profil temporel d’activité. Pour l’évaluation présente et future du risque de piqûre par I. ricinus, des modèles météo-sensibles de prédiction de l’activité sont nécessaires. Dans cette thèse un modèle météo-sensible de régression linéaire généralisé a été ajusté sur des séries temporelles de captures de nymphes, acquises antérieurement à la thèse, pour prédire l’activité saisonnière des nymphes. Depuis ce modèle des profils saisonniers ont été simulés sous scénario de changement climatique. Un modèle de survie météo-sensible a été ajusté à des durées de survie chez des adultes femelles I. ricinus dans un cadre bayésien. Simultanément à la modélisation, nous avons investigué les profils d’activité saisonnière des nymphes I. ricinus sur plusieurs observatoires répartis dans les climats de France métropolitaine par des captures mensuelles en continu d’avril 2014 à juillet 2016. Le comportement d’I. ricinus sous des conditions informatives pour le changement climatique a été investigué. Nos observations et nos simulations confortent l’hypothèse d’une augmentation progressive de l’activité d’I. ricinus sur la saison d’hiver, avec des pics d’activité plus précoces. Une activité modérée a été mise en évidence sous des vagues de chaleur à certains moments du jour, suggérant l’importance de l’échelle infra-journalière en été. Le modèle de survie a reproduit avec succès l’effet très défavorable des conditions sèches sur la survie des femelles. Notre travail de thèse souligne l’importance i) d’acquérir des observations au plus proche des futures conditions saisonnières, ii) de renforcer la sensibilité des modèles de dynamique de populations à la disponibilité en eau dans le milieu des tiques, iii) d’étudier les effets de la météorologie vécue sur la population à l’échelle inter‑annuelle. / The tick species Ixodes ricinus is a vector of major concern for animal and public health in Europe. Climate change will probably modify its activity time patterns. In the present and future bite risk assessment, weather-based models are required to predict tick activity. In this work a weather-based generalized linear regression model was fitted to tick count time series to predict the seasonal activity in I. ricinus nymphs. Using this model we simulated activity time series with climate change weather time series as inputs. A weather-based model was fitted to survival durations in I. ricinus females using a Bayesian estimation method. Simultaneously to modeling, we settled several observatories located in the climate areas encountered in mainland France to investigate the seasonal activity patterns of I. ricinus populations. Monthly tick collection was performed in routine from April 2014 to July 2016. I. ricinus behavior was investigated under conditions which are informative to assess the impact of climate change on activity. Both our observed and simulated results reinforce the hypothesis that activity periods will spread over winter under climate change. In summer season, activity was moderately high at some moments of the day, which underlined that the intra-day scale should be considered when assessing summer activity. The survival model successfully simulated the critical effect of dryness on female survival. In this work we highlighted the need i) to acquire observations under present conditions which are close to the ones projected under climate change, ii) to strengthen the sensitivity of population dynamic models to water availability in the close environment of ticks, and iii) to investigate the effects of the weather conditions undergone by ticks on their population dynamics at the inter-annual scale.