<div>Wildlife diseases are an increasingly growing concern for public health managers, conservation biologists, and society at large. These diseases may be zoonotic -- infective wildlife are able to spread pathogens to human populations. Animal or plant species of conservation concern may also be threatened with extinction or extirpation due to the spread of novel pathogens into their native ranges. In this thesis, I develop some mathematical methods for understanding the dynamics of vector-borne diseases in wildlife populations which include several elements of host and vector biology. </div><div><br></div><div>We consider systems where a vector-borne pathogen is transmitted to a host population wherein individuals either die to disease or recover, remaining chronically infective. Both ordinary differential equations (ODE) and individual based (IBM) models of such systems are formulated then applied to a specific system of wildlife disease: avian malaria in Hawaiian honeycreeper populations -- where some species endure disease-induced mortality rates exceeding 90\%. The ODE model predicts that conventional management methods cannot fully stop pathogen transmission.</div><div><br></div><div>Vector dispersal and reproductive biology may also play a large role in the transmission of vector-borne diseases in forested environments. Using an IBM which models dispersal and mosquito reproductive biology, we predict that reducing larval habitat at low elevations is much more effective than at higher elevations. The ODE model is extended to include distinct populations of sensitive and tolerant hosts. We find that the form which interaction between the hosts takes has a significant impact on model predictions.</div>

Background Dengue is a significant problem of international health concern. According to the World Health Organization in 2012, globally, dengue is “the most important mosquito borne viral disease” with incidence 30 higher than it had been 50 years ago. While most of the burden of disease associated with dengue is located in areas with a tropical and sub-tropical climate, increasing evidence suggests temperate areas are also at risk. Considering the recent introduction of relevant mosquito vectors into Southern Europe, and increasing numbers of imported dengue via travelers, Europe and other temperate areas may be increasingly at risk for dengue emergence, establishment and local transmission in the foreseeable future. Methods Recent dengue emergence in Madeira and reemergence in Tokyo underline the hypothesis that passenger air-travel can be an important conduit for the importation of vector-borne disease leading to emergence in naïve areas climatically suitable for dengue transmission, including parts of Europe. Combining information on travel with virus genetic similarity was useful in discerning likely pathways of for the importation of infections. Generalizing information learned from outbreaks in Tokyo and Madeira with global epidemic intelligence, global travel networks, and climate change projections, leads to more refined understanding of the magnitude of dengue infectious imported into temperate areas and these virus introduction events’ potential implications for seeding epidemics in the 21st century. Results While compared to total travel, imported dengue events and epidemics of dengue outside the tropics are rare, our combined evidence and modeled estimations suggest strongly that epidemic dengue emergence in temperate areas is possible and will continue to increase. We found that global change dynamics including warming temperatures in the much of the northern hemisphere and increasing passenger interconnectivity between areas endemic for dengue and dengue free areas are key mechanisms partly explaining these unprecedented epidemiological transitions. Conclusion While we calibrated our models on information known about dengue, many elements of the methods and conclusions may increase understanding of the potentially global implications for imported infections of other climate-sensitive infectious diseases’ that may have similar parameters. During 2016 and the years to come, techniques developed in this doctoral research will contribute to models used in risk analysis for vector-borne diseases of interest, including the increasing important potential for imported Chikungunya and Zika viruses into a variety of unexposed areas.

Diseases such as malaria and dengue impose great economic burdens and are a serious threat to public health, with young children being among the worst affected. These diseases are transmitted by mosquitoes, also called disease vectors, which are able to transmit both parasitic and viral infections. One of the most important strategies in the battle against mosquito-borne diseases is vector control by insecticides and the goal is to prevent people from being bitten by mosquitoes. Today’s vector control methods are seriously threatened by the development and spread of insecticide-resistant mosquitos warranting the search for new insecticides. This thesis has investigated the possibilities of vector control using non-covalent inhibitors targeting acetylcholinesterase (AChE); an essential enzyme present in mosquitoes as well as in humans and other mammals. A key requirement for such compounds to be considered safe and suitable for development into new public health insecticides is selectivity towards the mosquito enzyme AChE1. The work presented here is focused on AChE1 from the disease transmitting mosquitoes Anopheles gambiae (AgAChE1) and Aedes aegypti (AaAChE1), and their human (hAChE) and mouse (mAChE) counterparts. By taking a medicinal chemistry approach and utilizing high throughput screening (HTS), new chemical starting points have been identified. Analysis of the combined results of three different HTS campaigns targeting AgAChE1, AaAChE1, and hAChE allowed the identification of several mosquito-selective inhibitors and a number of compound classes were selected for further development. These compounds are non-covalent inhibitors of AChE1 and thereby work via a different mechanism compared to current anti-cholinergic insecticides, whose activity is the result of a covalent modification of the enzyme. The potency and selectivity of two compound classes have been explored in depth using a combination of different tools including design, organic synthesis, biochemical assays, protein X-ray crystallography and homology modeling. Several potent inhibitors with promising selectivity for the mosquito enzymes have been identified and the insecticidal activity of one new compound has been confirmed by in vivo experiments on mosquitoes. The results presented here contribute to the field of public health insecticide discovery by demonstrating the potential of selectively targeting mosquito AChE1 using non-covalent inhibitors. Further, the presented compounds can be used as tools to study mechanisms important in insecticide development, such as exoskeleton penetration and other ADME processes in mosquitoes.

The history of mitigation programs to contain vector-borne diseases is a story of successes and failures. Due to the complex interplay among multiple factors that determine disease dynamics, the general principles for timely and specific intervention for incidence reduction or eradication of life-threatening diseases has yet to be determined. This research discusses computational methods developed to assist in the understanding of complex relationships affecting vector-borne disease dynamics. A computational framework to assist public health practitioners with exploring the dynamics of vector-borne diseases, such as malaria and dengue in homogenous and heterogeneous populations, has been conceived, designed, and implemented. The framework integrates a stochastic computational model of interactions to simulate horizontal disease transmission. The intent of the computational modeling has been the integration of stochasticity during simulation of the disease progression while reducing the number of necessary interactions to simulate a disease outbreak. While there are improvements in the computational time reducing the number of interactions needed for simulating disease dynamics, the realization of interactions can remain computationally expensive. Using multi-threading technology to improve performance upon the original computational model, multi-threading experimental results have been tested and reported. In addition, to the contact model, the modeling of biological processes specific to the corresponding pathogen-carrier vector to increase the specificity of the vector-borne disease has been integrated. Last, automation for requesting, retrieving, parsing, and storing specific weather data and geospatial information from federal agencies to study the differences between homogenous and heterogeneous populations has been implemented.

Depuis les années 1970, les pays du Pacifique sont de plus en plus fréquemment touchés par des maladies vectorielles telles que la Dengue, le Chikungunya ou le Zika. Le contrôle de ces maladies nécessite la connaissance de leur distribution spatio-temporelle au sein de la population ainsi que la compréhension des facteurs et mécanismes, souvent multiples, régissant cette distribution. Dans cette thèse, nous nous intéressons à la modélisation spatio-temporelle des déterminants de la dynamique spatiale de la dengue à l'échelle régionale du Pacifique, l'échelle territoriale de la Nouvelle-Calédonie, et l'échelle d'une ville, Nouméa, capitale de la Nouvelle-Calédonie.Dans le Pacifique, la dengue survient sous la forme de vagues épidémiques successives dues à l'introduction et à la diffusion régionale d'un nouveau sérotype tous les cinq à sept ans. En Nouvelle-Calédonie, la dengue présente une dynamique épidémique saisonnière, le sérotype dominant étant celui circulant dans la région. L'émergence d'une épidémie nécessite des conditions climatiques précises, et un indicateur annuel prédictif du risque d'émergence est maintenant utilisé de manière opérationnelle par les autorités de santé. Sur le plan spatial, au cours d'une épidémie, en moyenne, la circulation du virus est plus intense dans les zones où la température moyenne ainsi que les densités locales de population sont élevées. Que ce soit sur le territoire entier ou dans la ville de Nouméa, lors de la ré-émergence d'un même sérotype, la diffusion spatiale du virus paraît limitée par l'immunité de population créée par les épidémies précédentes. Cette thèse permet de mettre en évidence la nature complexe et multi-factorielle des maladies vectorielles, et de souligner l'intérêt d'analyses multi-échelles pour l'étude de leur épidémiologie. Au-delà des résultats obtenus sur la dengue dans la région Pacifique, notre volonté était de développer un cadre méthodologique pour l'analyse spatio-temporelle des données de surveillance épidémiologique applicable à d'autres contextes géographiques ou épidémiologiques. / Since the 1970's, the frequency of vector-borne diseases such as Dengue, Chikungunya or Zika has significantly increased in the Pacific region. Understanding the factors and mechanisms underlying the spatio-temporal distribution of these diseases provides useful information regarding their control and prevention. In this thesis, we identified dengue spatio-temporal patterns and used modeling tools to identify the factors associated to an increased epidemiological risk at a regional scale (Pacific), a territorial scale (New-Caledonia), and a city scale (Noumea, the capital of New-Caledonia).Every five to seven years, dengue spreads over the entire Pacific as large epidemics caused by the introduction and regional diffusion of one of the four dengue virus serotypes. In New Caledonia, dengue has a seasonal epidemic pattern. The emergence of an epidemic requires specific climatic conditions. The identification of these conditions led to the implementation of an operational early warning system to predict dengue annual epidemic risk. Spatially, at the territorial scale, during epidemic years, high levels of viral circulation are found in areas with higher mean temperature and higher local population densities. Whether at the territorial scale or at the city scale, the spatial diffusion of the virus during epidemics caused by the re-emergence of the same serotype seems limited by the population immunity created by past epidemics. This thesis highlights the complexity and the multi-factorial aspect of vector-borne diseases, and discusses the usefulness of a multi-scale approach in modelling their epidemiology. Besides enhancing our understanding of dengue epidemiology over the Pacific area, we also developed a methodological framework that can be used in other geographical or epidemiological settings for the spatio-temporal analysis and modeling of epidemiological surveillance data.

En outre, nous avons fait une expérience sur les nouveaux outils pour identifier les tiques par MALDI-TOF MS protéines profilage et des méthodes moléculaires. Notre étude visant à explorer les bactéries dans les ixodidae prélevés sur des animaux domestiques en Éthiopie a révélé une prévalence globale de 6% (46/767) des rickettsies de SFG, 3,8% (29/767) ADN de Borrelia et 6,4% (54/842) de C. burnetii dans différentes espèces de tiques. L'étude pour étudier les bactéries dans 303 puces prélevés sur des chiens et des chats domestiques en Ethiopie qui ont été identifiés comme étant morphologiquement Ctenocephalides felis felis, Ctenocephalides canis, Pulex irritans et Echidnophaga gallinacé montré Rickettsia felis dans 21% des puces, principalement dans Ctenocephalides felis, avec un semblable prévalence dans les puces de chiens et de chats. La présence d'Acinetobacter spp. dans M. ovinus, Heterodoxus spiniger, Bovicola ovis et Linognathus vituli. La séquence du gène rpoB partiel a révélé la présence de A. soli, A. lowffii, A. Pitti et 3 nouveaux Acinetobacter spp. dans les poux et Keds. Bartonella melophagi a été identifié par une PCR standard, suivi par un séquençage du fragment de la gltA et gène rpoB chez M. ovinus. Dans l'ensemble, nos résultats alerte les médecins en charge des patients avec fièvre d'étiologie inconnue en Ethiopie et ceux qui se soucient de voyageurs en provenance de l'Ethiopie à prendre en compte la présence de plusieurs espèces zoonotiques à transmission vectorielle de bactéries, y compris SFG rickettsies, C. burnetii, R. felis, B. henselae et B. melophagi comme agents pathogènes potentiels. / Our study to explore bacteria in ixodid ticks collected from domestic animals in Ethiopia revealed an overall prevalence of 6% (46/767) SFG rickettsiae, 3.8% (29/767) Borrelia DNA and 6.4% (54/842) C. burnetii in different tick species. The study to investigate bacteria in 303 fleas collected from domestic dogs and cats in Ethiopia that were morphologically identified as Ctenocephalides felis felis, Ctenocephalides canis, Pulex irritans and Echidnophaga gallinacean showed Rickettsia felis in 21% of fleas, mainly in Ctenocephalides felis, with a similar prevalence in fleas from dogs and cats. The study to investigate bacteria in lice and sheep ked (Melophagus ovinus) revealed Acinetobacter spp. in M. ovinus, Heterodoxus spiniger, Bovicola ovis and Linognathus vituli. Partial rpoB gene sequence revealed A. soli, A. lowffii, A. pitti and 3 new Acinetobacter spp. in the lice and keds. Molecular identification of lice using an 18S rRNA gene analysis confirmed the morphological methods of lice identification. Bartonella melophagi was identified by standard PCR followed by sequencing of fragments of the gltA and rpoB genes in M. ovinus.Overall, our findings alert physicians managing patients with fever of unknown aetiology in Ethiopia and those who care for travellers from Ethiopia to consider the presence of several vector-borne zoonotic species of bacteria including SFG rickettsiae, C. burnetii, R. felis, B. henselae and B. melophagi as potential causative agents.

L'évolution de la résistance aux insecticides chez les moustiques responsables de la transmission de maladies infectieuses compromet notre capacité à contrôler ces populations de vecteurs et pose de graves problèmes de santé publique. Mais les nombreuses modifications physiologiques associées au phénomène de résistance aux insecticides pourraient altérer l'épidémiologie de ces maladies de manière plus indirecte en modifiant la capacité vectorielle des moustiques. Afin d'étudier cette question nous avons mis en place un nouveau système expérimental composé du parasite aviaire Plasmodium relictum SGS1 et de son vecteur naturel le moustique Culex pipiens. Nous avons étudié l'effet de différents allèles de résistance aux insecticides (représentant deux mécanismes principaux i.e. la résistance métabolique ou la modification de la cible) sur une série de traits d'histoire de vie du parasite et du moustique. L'impact de ces différents allèles a été étudié d'une part, dans les conditions contrôlées de leur expression dans un même fond génétique (en utilisant plusieurs souches de moustiques isogéniques), et d'autre part, dans les conditions plus réalistes de leur expression dans un fond génétique hétérogène (utilisation de moustiques échantillonnés sur le terrain). Nous montrons que la résistance aux insecticides a des effets pleïotropes sur l'immunocompétence et les traits d'histoire de vie des moustiques. Son effet sur le développement de Plasmodium semble en revanche limité. Nous discutons d'une part, de la nécessité de poursuivre une approche multifactorielle (impliquant la physiologie, l'immunité et le comportement des moustiques) afin de mieux comprendre l'impact de la résistance aux insecticides sur la transmission de Plasmodium, et d'autre part des perspectives intéressantes qu'offrent ce nouveau système expérimental pour l'étude de l'écologie évolutive des maladies à vecteurs. / The evolution of insecticide resistance in mosquitoes threatens our ability to control many-vector-transmitted diseases, thereby raising serious public health issues. Insecticide resistance entails numerous physiological changes in mosquitoes. This thesis investigates whether these physiological changes alter the quality of mosquitoes as vectors of malaria. To address this issue, we developed a new experimental system consisting in the avian malaria parasite Plasmodium relictum SGS1 and its natural vector, the mosquito Culex pipiens. We investigated the impact of two insecticide resistance mechanisms (target site resistance and metabolic resistance) on several mosquito and parasite life history traits relevant for malaria transmission. The effect of different insecticide resistant genes was investigated using both isogenic laboratory mosquito strains (i.e. against a homogeneous genetic background) and sympatric field caught mosquitoes (i.e. under the more realistic, albeit noisier, conditions of a heterogeneous genetic background). We show that insecticide resistance has a pleiotropic effect on several mosquito traits (immunocompetence, longevity, fecundity), whereas it has only a limited effect on Plasmodium development. We discuss, on the one hand, the need to pursue such a multi-factorial approach (combining the mosquito physiology, immunity and behavior) to better understand the impact of insecticide resistance on malaria transmission and, on the other hand, the promising perspectives offered by this new experimental system for studying the evolutionary-ecology of infectious diseases.

Eviter la piqûre de moustiques vecteurs en utilisant des mesures antivectorielles reste le meilleur moyen de se protéger des maladies vectorielles. La salive de moustique peut induire une réponse anticorps (Acs) spécifique chez l’hôte qui pourrait être utilisé pour définir l'efficacité de ces mesures de protection antivectorielle. L’objectif de notre projet était d’évaluer la possibilité d’utiliser cette réponse Acs anti-salive de moustiques pour mesurer l’exposition à des espèces spécifiques de moustiques ainsi que d’identifier des marqueurs d’exposition. Nous nous sommes tout d’abord assurés de l’absence de différences intraspécifiques entre différentes colonies de moustiques, une condition indispensable pour pouvoir observer des différences au niveau de l’espèce. Par ailleurs, nous avons mis au point un protocole pour préserver les échantillons salivaires dans des conditions de terrains non optimales. A partir de ces expérimentations préliminaires, nous avons évalué la diversité du répertoire protéique salivaire de quatre espèces d’Anopheles par des différentes approches, et montré une spécificité de genre et d’espèce aussi bien au niveau protéique qu’antigénique. Enfin, nous avons montré une évolution spatio-temporelle de l’intensité de la réponse Acs anti-salive ainsi que sa spécificité de genre et d’espèce, chez des individus exposés à différents niveaux à Ae. caspius. Ces résultats souligne la possibilité de caractériser des antigènes salivaires spécifiques de genre et d’espèces qui peuvent avoir un intérêt pour mesurer le contact hôte/vecteur au niveau individuel, le risque de transmission de maladies vectorielles ou l’efficacité des mesures antivectorielles. / The primary mean to protect individuals from arthropod-borne diseases is the prevention of bites from infected arthropods which could be achieved by vector control strategies. Mosquito saliva could induce a specific antibody response in exposed individuals that could be used to assess the effectiveness of anti-vector measures. The aim of this study is to assess the possibility to use anti-mosquito saliva antibody responses in order to evaluate the exposure to specific species of vectors and to identify salivary protein candidates that can be used as immunological markers of exposure. We first verify the lack of intraspecific differences among several mosquito colonies which is essential to further observe potential differences at the species level. Moreover, a convenient storage method was developed to preserve salivary samples in non optimal condition on the field. Based on these preliminary results, we evaluated the salivary gland protein repertory diversity among four Anopheles species using complementary approaches and we shown a genus and species specificity at the protein and antigen level. At least, a spatio-temporal evolution of anti-saliva antibody responses was shown according to the Aedes caspius density using sera of differentially exposed individuals. The specificity of this response was also reported at the genus and species level. All together, these results suggest the feasibility to characterize genus and species specific salivary antigens which could be used as immunological markers of exposure to evaluate host/vector contacts, the risk of vector-borne disease transmission or the effectiveness of anti-vector strategies.

<p>  </p> <p>Vector-borne diseases (VBDs) represent a significant health burden worldwide, threatening approximately 80% of the global population. Insecticide-based vector control is the most effective method to manage many VBDs, but its efficacy has been declining due to high levels of resistance in vector populations to the main insecticide classes which operate via limited modes of action. Therefore, the discovery of new chemistries from non-conventional chemical classes and with novel modes of action is a priority for the control of vectors and VBDs. Natural products (NPs) are diverse in chemical structures and, potentially, modes of action. They have been used as insecticides for many decades and have inspired the development of multiple synthetic insecticides, suggesting the discovery of novel NPs could lead to the development of highly effective insecticides. </p> <p><br></p> <p>In this thesis, I report two studies with a main goal to identify novel mosquito-active insecticide leads that operate via modes of action distinct from existing insecticides. First, I tested the hypothesis that new mosquito-active insecticide leads with novel chemical structures, possibly operating via novel modes of action, can be identified by high-content larval phenotypic screening against a natural product collection and using novel phenotypic endpoints in addition to mortality endpoints. Here, I performed a high-content larval phenotypic screen using first instar (L1) larvae of <em>Aedes aegypti</em> (Linnaeus, 1762) against 3,680 compounds from the AnalytiCon MEGx Natural Product Libraries and a screening platform developed by Murgia et al., (2022). Compounds were screened in a 384-well plate format using the Perkin Elmer Opera Phenix and larvae were scored for lethal and novel phenotypic endpoints. Screening revealed five chemistries that caused larval mortality, including rotenone and a new NP chemistry, NP-4. The identification of rotenone confirmed the ability of the screen to detect mosquito-active NP chemistries. NP-4 caused high levels of larval mortality in the screen, and toxicity was confirmed in a subsequent concentration-response assay against third instar (L3) larvae of <em>Ae. aegypti</em>. 140 chemistries that caused atypical larval phenotypes, including cuticular pigmentation and morphometric changes relative to negative controls, were also identified by the screen. Some of these chemistries may operate by disruption of pathways regulating melanization, growth and development, and novel targets in the insect nervous systems, thus representing potential leads for further insecticide toxicity and mode of action studies. To facilitate quantitative analyses of atypical phenotypes, an attempt was made to assess the morphometrics of the thorax in larvae exposed to test chemistry, relative to control larvae. However, assessment was limited by the number of larvae images of suitable quality for measurements. </p> <p><br></p> <p>In the second study, I tested the hypothesis that metergoline (Murgia et al., 2022) and NP-4 (this study), two chemistries identified by the HTP phenotypic screen described in this project, operate via disruption of targets in the insect nervous systems that are distinct from the current insecticidal modes of products used in mosquito control programs. Specifically, I explored the hypothesis that metergoline operates via one or more insect orthologs of the mammalian G protein-coupled serotonin and dopamine receptors. An electrophysiology study was performed using the suction electrode technique and ganglia of the German cockroach, <em>Blattella germanica </em>(Linnaeus, 1767). To facilitate the investigation of metergoline agonism/antagonism and disruption of invertebrate GPCR signaling, 5-hydroxytryptamine (5-HT; serotonin) was included as a chemical probe. Electrophysiological recordings showed 5-HT (10µM and 1mM) and metergoline (10µM) caused no significant neurological activity at the tested concentrations in comparison to the saline control. However, a consistent neuro-inhibitory trend was observed, suggesting possible agonism of a 5-HT1-like receptor ortholog and antagonism of a putative 5-HT7-like receptor ortholog in the cockroach, respectively.  NP-4 caused significant neuro-inhibition at the tested concentration of 20µM, in comparison to the negative saline control. Given the demonstration of rapid contact toxicity to <em>Ae. aegypti</em> larvae and neurological inhibition in <em>B. germanica</em>, we propose NP-4 may act at one or more conserved targets in the insect nervous systems, which remain to be elucidated. </p> <p><br></p> <p>The significance of the present study is three-fold. First, this study reports the first high-content phenotypic screen of mosquito larvae against a NP collection and identification of 145 mosquito-active chemistries associated with lethal and phenotypic endpoints. These data confirm that the screening platform provided an innovative and effective system to rapidly identify mosquito-active small molecules with potential novel modes of action. Second, metergoline and NP-4 represent potential novel chemical leads for the development of new insecticides that can be incorporated into vector control programs targeting insecticide-resistant populations. Lastly, the study describes the first electrophysiology study of 5-HT, metergoline, and NP-4 via the suction electrode technique in an insect system and contributes new knowledge to the study of the insect serotonergic system, which represents an expanding area of vector biology research given its roles in feeding regulation.  </p> <p><br></p> <p>Future studies resulting from this thesis might include: (1) development of a set of morphometric criteria for quantitative analyses of atypical larval phenotypes, (2) incorporation of new phenotypic endpoints to expand the capacity of the screen to identify novel mode of action chemistries for insecticide discovery, and (3) identification of chemistry candidates suitable for further development from the 140 chemistries associated with atypical larval phenotypes in the primary screen using chemo-informatic and toxicological studies. In addition, studies using reverse transcription-polymerase chain reaction (RT-PCR), cell-based expression systems, mutant/insecticide resistant strains, and patch clamp electrophysiology could be pursued to further investigate the molecular mode of action of metergoline and NP-4, and potential for vector control.</p>

Bed bugs (Hemiptera:Cimicidae) are a common, hematophagous ectoparasite of humans and other animals, and are experiencing an international resurgence. Cimicids have been implicated in the transmission many disease agents, including various Bartonella species, however disease transmission has not yet been confirmed. Bartonella spp. are transmitted by a variety of arthropods, including fleas, lice and sand flies, and it is speculated that bed bugs may also serve as a potential vector. In this study, we used an artificial membrane to feed two groups of adult Cimex lectularius rabbit blood, either infected or uninfected with Bartonella henselae. After two weeks, the presence of Bartonella henselae was assessed via PCR, and salivary glands from infected and uninfected bed bugs were dissected and processed for transmission electron microscopy. We were unable to visually identify Bartonella henselae in the images, and therefore unable to confirm the role of bed bugs in B. henselae transmission.