Pas de résumé français / Each Bartonella species appears to be highly adapted to one or a limited number of reservoir hosts, in which it establishes a long-lasting intraerythrocytic bacteremia as the hallmark of infection. Although the course of Bartonella infection has been precisely described, the molecular mechanisms of host specific erythrocyte infection and the stages of precedent the arrival in the bloodstream are poorly understood. In this thesis we purposed to identify the mechanisms of erythrocyte infection by Bartonella and characterize the possible locations of Bartonella during the days before the intraerythrocytic stage.By the establishment of an in vitro model of adhesion and invasion of erythrocytes by Bartonella spp., we demonstrated that host specificity was determined by the interaction between bacteria and erythrocytes. By screening signature-tagged mutagenesis (STM) library of B. birtlesii in vivo and in vitro and ectopic expression, we revealed that type IV Trw locus was required for host-restricted adhesion to erythrocytes in a wide range of mammals. After that, we further characterized that only TrwJ1 and TrwJ2 were expressed and present on the surface of the bacteria and had the ability to bind to mouse erythrocytes, and the receptor of them was erythrocyte band3 by different technology (phage display, electron microscopy, far western blot and adherence and invasion inhibition assay). By the model of experimental infection of laboratory normal Balb/C mice and splenectomized mice with B. birtlesii, we showed that during the first 7 days, no bacteria were recovered from lymph nodes, bone marrow and brain, but in the spleen, transient in the liver, And bacteremia was the same in both infection models during the first 7 days, thereafter, bacteremia was 10 fold higher in splenectomized mice than in normal mice and lasted 2 weeks longer. This suggested that the spleen was able to retain Bartonella.In conclusion, the host specific adhesion between Bartonella and erythrocyte was mediated by Trw and erythrocytic band 3, and spleen had a role in retention Bartonella.

Leishmaniasis is a disease circulating in endemic areas between sand flies (Diptera: Phlebotominae) and reservoir hosts, which - in the case of Leishmania major - are principally rodents (Rodentia). Unlike in human patients, leishmaniasis is often asymptomatic in animal hosts. For transmission and maintenance of the parasite in nature, infectiousness of hosts for sand flies is essential; and the only method to directly test the infectiousness is xenodiagnosis. The main objective of this thesis is to establish a laboratory model for studying xenodiagnosis with L. major on inbred BALB/c mice and then to apply this model to potential reservoir ro- dents from the genus Mastomys. BALB/c mice were infected by intradermal inoculation of infective stages of L. major (iso- lated from sand fly guts) together with salivary gland homogenates from Phlebotomus duboscqi; infected mice were then exposed to P. duboscqi females for a period of ten weeks. Two inbred lines of BALB/c mice differed significantly in both the manifestation of the disease and infectiousness for sandflies. In BALB/c OlaHsdmice, great lesions were formed (up to 10 mm), mice were able to infect sand flies from the 2nd week after infection and their infec- tiousness reached up to 20.1% during the experiment. In BALB/c AnNCrl mice, only small...

A cutaneous leishmaniasis is the most common clinical form of human disease caused by parasite of the genus Leishmania. They are transmitted between the hosts by haematophagous females of dipteran sand flies of the genus Phlebotomus in the Old World and Lutzomyia in the New World. One of the major agents of cutaneous leishmaniasis in the Old World is Leishmania major. The disease caused by this species is a zoonosis where rodents act as reservoir host. The parasite long time circulates between reservoir rodents and sand flies, while humans are infected only accidentaly in the focus of infection. Rodents of the genus Arvicanthis belongs to the most abundant in the African continent. The genus has evolved in Ethiopia from where it expanded to a major part of Sub - Saharan Africa and the delta of the river Nile. These rodents are very abundant in endemic locations of cutaneous and visceral leishmanias and fulfil many reservoir host criterias including repeated field findings of individuals infected by L. major and another Leishmania species in nature. However, their role in the disease cycle remains to be confirmed. A. neumanni used in this study is an East African species spread from Ethiopia and Somalia to Kenya and Tanzania. Animals were experimentally infected with three different L. major...

Background: In order to optimize net transmission success, parasites are hypothesized to evolve towards causing minimal damage to their reservoir host while obtaining high shedding rates. For many parasite species however this paradigm has not been tested, and conflicting results have been found regarding the effect of arenaviruses on their rodent host species. The rodent Mastomys natalensis is the natural reservoir host of several arenaviruses, including Lassa virus that is known to cause Lassa haemorrhagic fever in humans. Here, we examined the effect of three arenaviruses (Gairo, Morogoro and Lassa virus) on four parameters of wild-caught Mastomys natalensis: body mass, head-body length, sexual maturity and fertility. After correcting for the effect of age, we compared these parameters between arenavirus-positive (arenavirus RNA or antibody) and negative animals using data from different field studies in Guinea (Lassa virus) and Tanzania (Morogoro and Gairo viruses). Results: Although the sample sizes of our studies (1297, 749 and 259 animals respectively) were large enough to statistically detect small differences in body conditions, we did not observe any adverse effects of these viruses on Mastomys natalensis. We did find that sexual maturity was significantly positively related with Lassa virus antibody presence until a certain age, and with Gairo virus antibody presence in general. Gairo virus antibody-positive animals were also significantly heavier and larger than antibody-free animals. Conclusion: Together, these results suggest that the pathogenicity of arenaviruses is not severe in M. natalensis, which is likely to be an adaptation of these viruses to optimize transmission success. They also suggest that sexual behaviour might increase the probability of M. natalensis to become infected with arenaviruses.

Lyme borreliosis is a tick-transmitted infectious disease. The causative agents are spiral-shaped bacteria and the most common sign of infection is a skin rash at the site of the tick bite. If not treated with antibiotics, the bacteria can disseminate and cause a variety of different manifestations including arthritis, carditis or neurological problems. The disease is a zoonosis and the bacteria are maintained in nature by different vertebrate reservoir host animals. In Europe, three different Borrelia genospecies cause Lyme borreliosis: B. burgdorferi, B. afzelii and B. garinii. The latter depends in part on birds as its reservoir host. B. garinii bacteria have been found in a marine enzootic infection cycle worldwide and also among terrestrial birds. This thesis suggests that passerine birds and seabirds constitute an important reservoir for B. garinii bacteria also with clinical importance. We have found bacteria very similar to Lyme borreliosis causing isolates in ticks infesting migrating passerine birds. The birds not only transport infected ticks, but are competent reservoir hosts, as measured by their ability to infect naïve ticks. Their role as a reservoir host is dependent on their foraging behavior, where ground-dwelling birds are of greater importance than other species. When comparing B. garinii isolates from Europe, the Arctic and North Pacific, and including isolates from seabirds, passerine birds, Ixodes ricinus ticks and Lyme borreliosis patients, we found that phylogenetic grouping was not necessarily dependent on geographical or biological origin. B. garinii from seabirds were very heterogeneous and found in all different groups. Therefore, the marine and the terrestrial infection cycles are likely to overlap. This was supported by the fact that B. garinii isolated from seabirds can establish a long-term infection in mice. Bacteria from the genospecies B. garinii are overrepresented among neuroborreliosis patients. Interestingly, many clinical B. garinii isolates are sensitive to human serum and have shown weak binding to the complement inhibitor protein factor H. By transforming a serum-sensitive B. garinii isolate with a shuttle vector containing the gene for the factor H binding protein OspE from complement-resistant B. burgdorferi, serum resistance could be increased. In addition, neurovirulent B. garinii strains recently isolated from neuroborreliosis patients were shown to express a factor H binding protein, not found in bacteria that had been kept in culture for a long time. This protein may contribute to the virulence of neuroborreliosis-causing B. garinii strains. When testing B. garinii isolates from Lyme borreliosis patients and seabirds for resistance to human serum, all members of the latter group were sensitive to even low levels of serum. This suggests that seabird isolates are not capable of infecting humans. In agreement with this, B. garinii isolated from seabirds do not appear to bind human factor H.

La leptospirose est une zoonose bactérienne due à des Spirochètes du genre Leptospira. Elle est classée dans le groupe des maladies ré émergentes en raison du réchauffement climatique par l'OMS. Elle peut atteindre tous les mammifères et notamment l'Homme. On recense environ tous les ans 500 000 cas graves humains (c'est-à-dire ayant nécessité une hospitalisation) mais les difficultés du diagnostic laissent à penser que ce chiffre est sous évalué. La France métropolitaine est le pays d'Europe où le nombre de cas déclarés est le plus important (environ 300 / an). La contamination se produit après à un contact direct ou indirect d'une plaie ou de muqueuses saines avec les urines d'un animal infecté. Les hôtes peuvent être séparés en réservoirs, porteurs sains, et animaux malades ; les Rongeurs forment le réservoir le plus connu mais des représentants d'autres ordres de Mammifères, tels les Insectivores ou les Chiroptères, peuvent aussi jouer ce rôle. Le Ragondin (Myocastor coypus) est un grand Rongeur aquatique originaire d'Amérique du Sud qui a maintenant colonisé la quasi-totalité du territoire français. Cette espèce a été identifiée comme un porteur rénal potentiel de leptospires pathogènes en France au début des années 2000. Notre étude a été orientée suivant deux axes de travail. Tout d'abord un suivi de la contamination par les leptospires pathogènes de deux populations de Ragondin dans des zones humides de l'est de la France. Au cours de cette partie, nous nous sommes intéressés à la prévalence sérologique et à la prévalence du portage rénal dans chacune des populations. Cette étude a été complétée par une estimation de l'état général des animaux au cours d'autopsies détaillées et de l'évaluation histologique des reins afin de déterminer quel pouvait être l'impact, dans cette espèce, de l'infection par des leptospires pathogènes. Ces derniers résultats nous ont permis de clarifier, en partie, le rôle du Ragondin dans le cycle épidémiologique de la maladie. Dans un second axe, nous avons développé un outil permettant le suivi de la contamination des eaux environnementales par les leptospires pathogènes / Leptospirosis is one of the most prevalent bacterial zoonosis. It is caused by Spirochetes of the Leptospira genus and affects all mammals. The WHO considers it as a re emerging disease because of global warming. About 500 000 severe human cases are recognized worldwide but the incidence is probably under evaluated because of the diagnosis difficulties. Metropolitan France is the most affected European country with about 300 human cases declared per year. Infection results from exposure to infected urines of carrier animals, either directly or indirectly via contaminated environment. Animal hosts can be classified as reservoir hosts, healthy carriers or accidental hosts that suffer from the disease. Rodents are well known reservoir hosts but other mammals, as Insectivorous or Chiropters, can also be reservoirs for pathogenic leptospires. The nutria (Myocastor coypus) is a large aquatic rodent originating from South America. The species is now settled throughout France and it has been identified as a potential carrier of leptospirosis in 2001 in Western France. This work had two major goals. Firstly, we studied the contamination of two coypus’ populations by pathogenic leptospires. In this part we determined the serological prevalence and the prevalence of kidney carriage in both populations. Simultaneously, detailed necropsy and kidney histology were performed in order to characterize the impact of leptospirosis infection in this species and thus to clarify the role of the coypu in the epidemiological cycle of leptospirosis. Secondly, we developed a tool to detect and quantify pathogenic leptospires in environmental water that can be used in water quality surveillance program

Leishmaniasis is a human and animal disease caused by digenetic parasites of the genus Leishmania, which is now divided into 4 subgenera - L. (Leishmania), L. (Viannia), L. (Sauroleishmania) and L. (Mundinia). Subgenus Mundinia was established in 2016 and consists of 5 species - L. enriettii and L. macropodum are parasites of wild mammals and L. martiniquensis, L. orientalis and unnamed L. sp. from Ghana are infectious to humans. Mundinia are geographically widely dispersed, their distribution covers all continents, except of Antarctica. Despite phlebotomine sand flies (Diptera: Psychodidae) also biting midges (Diptera: Ceratopogonidae) are supposed to be involved in transmission of these species, which is a unique feature for this subgenus. But there is little to no current information on natural reservoir hosts and vector species for any Mundinia species. In this thesis we tested possible vectors and potential model organisms (Guinea-pigs) and reservoir hosts of Mundinia species by experimental infections. We used 3 sand fly species sharing geographical distribution with respective Mundinia species and available in our laboratory for experimental infections. Sand flies from Australia had never been colonised so we used the permissive vector Lu. migonei for testing development of L. macropodum....

Campylobacter jejuni is the leading bacterial cause of gastrointestinal diarrheal disease in humans worldwide. This zoonotic pathogen has a complex epidemiology due to its presence in many different host organisms. The overall aim of this thesis was to explore the role of amoebae of the genus Acanthamoeba as an intermediate host and vector for survival and dissemination of C. jejuni. Earlier studies have shown that C. jejuni can enter, survive and replicate within Acanthamoebae spp. In this thesis, I have shown that C. jejuni actively invades Acanthamoeba polyphaga. Once inside, C. jejuni could survive within the amoebae by avoiding localization to degradative lysosomes. We also found that A. polyphaga could protect C. jejuni in acid environments with pH levels far below the range in which the bacterium normally survives. Furthermore, low pH triggered C. jejuni motility and invasion of A. polyphaga. In an applied study I found that A. polyphaga also could increase the survival of C. jejuni in milk and juice both at room temperature and at +4ºC, but not during heating to recommended pasteurization temperatures. In the last study we found that forty environmental C. jejuni isolates with low bacterial concentrations could be successfully enriched using the Acanthamoeba-Campylobacter coculture (ACC) method. Molecular genetic analysis using multilocus sequence typing (MLST) and sequencing of the flaA gene, showed no genetic changes during coculture. The results of this thesis have increased our knowledge on the mechanisms behind C. jejuni invasion and intracellular survival in amoebae of the genus Acanthamoeba. By protecting C. jejuni from acid environments, Acanthamoebae could serve as important reservoirs for C. jejuni e.g. during acid sanitation of chicken stables and possibly as vectors during passage through the stomach of host animals. Furthermore, Acanthamoeba spp. could serve as a vehicle and reservoir introducing and protecting C. jejuni in beverages such as milk and juice. Validation of the ACC method suggests that it is robust and could be used even in outbreak investigations where genetic fingerprints are compared between isolates. In conclusion, Acanthamoeba spp. are good candidates for being natural hosts and vectors of C. jejuni.

A. phagocytophilum, une bactérie transmise par les tiques, est responsable de l’anaplasmose granulocytaire, une maladie émergente qui infecte une large gamme de mammifères dont l’homme. Actuellement, la description des cycles épidémiologiques de cette bactérie est incomplète. L’objectif de cette thèse est de caractériser la diversité génétique d’A. phagocytophilum chez différentes espèces d’hôtes, afin de déterminer quelles espèces participent au même cycle épidémiologique. D’abord, nous avons caractérisé la diversité génétique d’A. phagocytophilum chez les animaux domestiques malades à l’aide d’une MLSA. Nous avons identifié trois groupes de génotypes infectant les bovins, dont un groupe est partagé avec les chevaux et les chiens, et un avec les chevreuils. Ensuite, nous avons recherché quelles espèces de tiques pouvaient transmettre la bactérie, et quels pouvaient être les réservoirs parmi les mammifères sauvages. En Camargue, un génotype au fort potentiel zoonotique a été identifié chez cinq espèces de tiques du genre Rhipicephalus, Dermacentor et Hyalomma. La prévalence chez des rongeurs suggère qu’ils peuvent être réservoirs, mais la présence de génotypes infectant les bovins chez les mulots est à vérifier. Enfin, la comparaison des génotypes obtenus chez les tiques et les chevreuils par séquençage 454, a montré que la contribution des chevreuils à l’infection des tiques était faible sur le site des Vallons de Gascogne. L’absence de rongeurs infectés sur ce site suggère que d’autres mammifères réservoirs sont présents. Cette étude montre la complexité des cycles d’A. phagocytophilum et l’intérêt des outils moléculaires. / A. phagocytophilum, a tick-borne bacterium, is responsible of the granulocytic anaplasmosis, an emerging disease that infects a large range of mammals including humans. Currently, the description of the epidemiological cycles of this bacterium is incomplete. The objective of this thesis was to characterize the genetic diversity of A. phagocytophilum in different host species to determine those involved in the same epidemiological cycle. First, we characterized the genetic diversity of A. phagocytophilum in sick domestic animals with a MLSA. We identified three groups of genotypes infecting cattle, including one group shared with horses and dogs, and another shared with roe deer. Then, we investigated what species of ticks can transmit the bacteria, and what wild mammals could be reservoirs. In Camargue, a genotype with high zoonotic potential was identified in five species of ticks of the genus Rhipicephalus, Dermacentor and Hyalomma. The prevalence in French rodents suggests that they may be reservoir hosts, but the presence of genotypes infecting cattle in rodents must be checked. Finally, comparing the bacterial genotypes in ticks and roe deer by 454 sequencing, showed that the contribution of the roe deer to tick infection was low in the site of “Vallons de Gascogne”. The absence of infected rodents in this location suggests that other reservoir mammals are present. This study demonstrates the complexity of the A. phagocytophilum cycle and the contribution of molecular tools.

Wenige Studien haben erste Hinweise darauf geliefert, dass die insektivore Fledermausart Mops condylurus ein natürliches Reservoir von Ebolaviren sein könnte. Im Rahmen dieser Doktorarbeit wurden weitere Hinweise gesucht, um die Bedeutung dieser Fledermausart als Reservoirwirt für Ebolaviren besser beurteilen zu können. Dafür wurden die Expressionslevel des Membranproteins Niemann-Pick C1 (NPC1), welches essentiell für den Eintritt von Ebolaviren in ihre Wirtszellen ist, in vitro durch konfokale Mikroskopie und Durchflusszytometrie charakterisiert. In dieser Arbeit generierte Primärzellkulturen aus 12 verschiedenen Organen von M. condylurus zeigten für die meisten Primärzellen deutlich niedrigere Expressionslevel als Kontrollzellen von Mensch, Affe oder einer europäischen Fledermaus. Die untersuchte Replikationskinetik von Ebola virus (EBOV) zeigte in allen Primärzellen von M. condylurus niedrigere Replikationsraten, die meistens mit den niedrigen NPC1 Rezeptor-Expressionsleveln korrelierten. Geringere Mengen von NPC1 könnten in vivo zur Virusreplikation auf niedrigerem Niveau beitragen. Desweiteren zeigten alle Primärzellen von M. condylurus eine hohe Toleranz gegenüber EBOV ohne Zelltot. Eine beobachtete Persistenz in Lungenprimärzellen könnte die intrinsische Fähigkeit widerspiegeln, dass Ebolaviren auch in vivo in dieser Fledermausart persistieren könnten. Mit den geringeren NPC1-Rezeptor-Expressionsleveln, der geringeren Virusreplikation, der hohen Toleranz gegenüber EBOV und der Etablierung von persistenten Infektionen in Primärzellen von M. condylurus wurden in vitro zusätzliche Hinweise gewonnen, die die Wahrscheinlichkeit dieser Fledermausspezies als ein mögliches natürliches Reservoir von Ebolaviren erhöht. Ergebnisse von zusätzlichen Temperaturversuchen lassen vermuten, dass die Heterothermie der Fledermäuse einen Schlüsselfaktor für die Toleranz von Ebolaviren in vivo darstellen und darüber hinaus an der Balance zwischen Viruskontrolle und Virusvermehrung beteiligt sein könnte. / Few studies provide first evidence that Mops condylurus, an insectivorous microbat, could be a natural reservoir for ebolaviruses. The aim of this thesis was to investigate indicators to determine the potential role of this bat species in the ecology of ebolaviruses. Therefore, the expression levels of the membrane protein Niemann-Pick C1 (NPC1), essential for the entry of ebolaviruses into their host cells, were characterized in vitro by confocal microscopy and flow cytometry. Our generated primary cell cultures from 12 different organs from M. condylurus showed significantly lower expression levels in most primary cells compared to control cells from human, monkey or a European bat. In most cases, lower Ebola virus (EBOV) replication rates in primary cells from M. condylurus, determined by RT-qPCR, correlated to lower NPC1 receptor expression levels. Low NPC1 receptor expression levels may contribute to decreased virus replication rates also in vivo. Additionally, all primary cells were highly tolerant to EBOV infections without cell death. The observed establishment of persistent EBOV infections in lung primary cells from M. condylurus may reflect the intrinsic ability to persist in vivo in this bat species. With lower NPC1 receptor expression levels, lower virus replication rates, high tolerance to EBOV infections and establishment of persistent infections in primary cells from M. condylurus, the in vitro experiments provided further evidence that this bat species is a potential reservoir of ebolaviruses. Results from additional temperature experiments suggest, that heterothermy of M. condylurus could be a key factor for tolerance to EBOV infections in vivo and be involved in balancing the level of virus replication.