Insight into cross-talk between intra-amoebal pathogens.

Abstract: Background: Amoebae are phagocytic protists where genetic exchanges might take place between amoeba-resistant bacteria. These amoebal pathogens are able to escape the phagocytic behaviour of their host. They belong to different bacterial phyla and often show a larger genome size than human-infecting pathogens. This characteristic is proposed to be the result of frequent gene exchanges with other bacteria that share a sympatric lifestyle and contrasts with the genome reduction observed among strict human pathogens.Results: We sequenced the genome of a new amoebal pathogen, Legionella drancourtii, and compared its gene content to that of a Chlamydia-related bacterium, Parachlamydia acanthamoebae. Phylogenetic reconstructions identified seven potential horizontal gene transfers (HGTs) between the two amoeba-resistant bacteria, including a complete operon of four genes that encodes an ABC-type transporter. These comparisons pinpointed potential cases of gene exchange between P. acanthamoebae and Legionella pneumophila, as well as gene exchanges between other members of the Legionellales and Chlamydiales orders. Moreover, nine cases represent possible HGTs between representatives from the Legionellales or Chlamydiales and members of the Rickettsiales order.Conclusions: This study identifies numerous gene exchanges between intracellular Legionellales and Chlamydiales bacteria, which could preferentially occur within common inclusions in their amoebal hosts. Therefore it contributes to improve our knowledge on the intra-amoebal gene properties associated to their specific lifestyle.

Conjugation by Mosquito Pathogenic Strains of Bacillus sphaericus

A mosquito pathogenic strain of Bacillus sphaericus carried out the conjugal transfer of plasmid pAMß1 to other strains of its own and two other serotypes. However, it was unable to conjugate with mosquito pathogens from three other serotypes, with B. sphaericus of other DNA homology groups or with three other species of Bacillus. Conjugation frequency was highest with a strain having an altered surface layer (S layer). Conjugal transfer of pAMß1 was not detected in mosquito larval cadavers. B. sphaericus 2362 was unable to mobilize pUB110 for transfer to strains that had served as recipients of pAMß1. These observations suggest that it is unlikely that genetically engineered B. sphaericus carrying a recombinant plasmid could pass that plasmid to other bacteria

Survey of Infectious Etiologies of Bovine Abortion during Mid- to Late Gestation in Dairy Herds.

Bovine abortion of unknown infectious etiology still remains a major economic problem. Thus, we investigated whether Brucella spp., Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Coxiella burnetii are associated with abortion and/or stillbirth in Tunisian dairy cattle. Using a pan-Chlamydiales PCR, we also investigated the role of Chlamydiaceae, Waddlia chondrophila, Parachlamydia acanthamoebae and other members of the Chlamydiales order in this setting. Veterinary samples taken from mid to late-term abortions from twenty dairy herds were tested. From a total of 150 abortion cases collected, infectious agents were detected by PCR in 73 (48.66%) cases, 13 (8.66%) of which represented co-infections with two infectious agents. Detected pathogens include Brucella spp (31.3%), Chlamydiaceae (4.66%), Waddlia chondrophila (8%), Parachlamydia acanthamoebae (5.33%), Listeria monocytogenes (4.66%) and Salmonella spp. (3.33%). In contrast, Campylobacter spp. and Coxiella burnetii DNA were not detected among the investigated veterinary samples. This demonstrates that different bacterial agents may cause bovine abortion in Tunisia. This is the first report suggesting the role of Parachlamydia acanthamoebae in bovine abortion in Africa. Further studies with a larger number of samples are necessary to confirm whether this emerging pathogen is directly linked to abortion in cattle.

Genomics of "chlamydia"-related bacteria

AbstractThe Chlamydiales order is an important bacterial phylum that comprises some of the most successful human pathogens such as Chlamydia trachomatis, the leading infectious cause of blindness worldwide. Since some years, several new bacteria related to Chlamydia have been discovered in clinical or environmental samples and might represent emerging pathogens. The genome sequencing of classical Chlamydia has brought invaluable information on these obligate intracellular bacteria otherwise difficult to study due to the lack of tools to perform basic genetic manipulation. The recent emergence of high-throughput sequencing technologies yielding millions of reads in a short time lowered the costs of genome sequencing and thus represented a unique opportunity to study Chlamydia-re\ated bacteria. Based on the sequencing and the analysis of Chlamydiales genomes, this thesis provides significant insights into the genetic determinants of the intracellular lifestyle, the pathogenicity, the metabolism and the evolution of Chlamydia-related bacteria. A first approach showed the efficacy of rapid sequencing coupled to proteomics to identify immunogenic proteins. This method, particularly useful for an emerging pathogen such as Parachlamydia acanthamoebae, enabled us to discover good candidates for the development of diagnostic tools that would permit to evaluate at larger scale the role of this bacterium in disease. Second, the complete genome of Waddlia chondrophila, a potential agent of miscarriage, encodes numerous virulence factors to manipulate its host cell and resist to environmental stresses. The reconstruction of metabolic pathways showed that the bacterium possesses extensive capabilities compared to related organisms. However, it is still incapable of synthesizing some essential components and thus has to import them from its host. Third, the genome comparison of Protochlamydia naegleriophila to its closest known relative Protochlamydia amoebophila revealed a particular evolutionary dynamic with the occurrence of an unexpected genome rearrangement. Fourth, a phylogenetic analysis of P. acanthamoebae and Legionella drancourtii identified several genes probably exchanged by horizontal gene transfer with other intracellular bacteria that might occur within their amoebal host. These genes often encode mechanisms for resistance to metal or toxic compounds. As a whole, the analysis of the different genomes enabled us to highlight a large diversity in size, GC percentage, repeat content as well as plasmid organization. The abundant genomic data obtained during this thesis have a wide impact since they provide the necessary bases for detailed investigations on countless aspects of the biology and the evolution of Chlamydia-related bacteria, whether in wet lab or by bioinformatical analyses.RésuméL'ordre des Chlamydiales est un important phylum bactérien qui comprend de nombreuses espèces pathogènes pour l'homme et les animaux, dont Chlamydia trachomatis, responsable du trachome, la cause majeure de cécité d'origine infectieuse à travers le monde. Durant ces dernières décennies, de nombreuses bactéries apparentées aux Chlamydia ont été découvertes dans des échantillons environnementaux ou cliniques mais leur éventuel rôle pathogène dans le développement de maladies reste peu connu. Ces bactéries sont des intracellulaires obligatoires car elles ont besoin d'une cellule hôte pour se multiplier, ce qui rend leur étude particulièrement difficile. Le développement de nouvelles technologies permettant de séquencer le génome d'un organisme rapidement et à moindre coût ainsi que l'essor des méthodes d'analyse s'y rapportant représentent une opportunité exceptionnelle d'étudier ces organismes. Dans ce contexte, cette thèse démontre l'utilité de la génomique pour développer de nouveaux outils diagnostiques ainsi que pour étudier le métabolisme de ces bactéries, leurs facteurs de virulence et leur évolution.Ainsi, une première approche a illustré l'utilité d'un séquençage rapide pour obtenir les informations nécessaires à l'identification de protéines qui sont reconnues par des anticorps humains ou animaux. Cette méthode, particulièrement utile pour un pathogène émergent tel que Parachlamydia acanthamoebae, a permis de découvrir de bons candidats pour le développement d'un outil diagnostique qui permettrait d'évaluer à plus large échelle le rôle de cette bactérie notamment dans la pneumonie. L'analyse du contenu génique de Waddlia chondrophila, un autre germe qui pourrait être impliqué dans les avortements et tes fausses-couches, a en outre mis en évidence la présence de nombreux facteurs connus qui lui permettent de manipuler son hôte. Cette bactérie possède de plus grandes capacités métaboliques que les autres Chlamydia, mais elle est incapable de synthétiser certains composants et doit donc les importer de son hôte pour subvenir à ses besoins. La comparaison du génome de Protochlamydia naegleriophila à son plus proche parent, Protochlamydia amoebophila, a dévoilé une évolution dynamique particulière avec l'occurrence d'un réarrangement majeur inattendu après la séparation de ces deux espèces. En outre, ces études ont montré l'occurrence de plusieurs transferts de gène avec d'autres organismes plus éloignés, notamment d'autres intracellulaires d'amibes, souvent pour l'acquisition de mécanismes de résistances à des composés toxiques. Les données génomiques acquises durant ce travail posent les fondements nécessaires a de nombreuses analyses qui permettront progressivement de mieux comprendre de nombreux aspects de ces bactéries fascinantes.

Short term evolution of a highly transmissible methicillin-resistant Staphylococcus aureus clone (ST228) in a tertiary care hospital.

Staphylococcus aureus is recognized as one of the major human pathogens and is by far one of the most common nosocomial organisms. The genetic basis for the emergence of highly epidemic strains remains mysterious. Studying the microevolution of the different clones of S. aureus is essential for identifying the forces driving pathogen emergence and spread. The aim of the present study was to determine the genetic changes characterizing a lineage belonging to the South German clone (ST228) that spread over ten years in a tertiary care hospital in Switzerland. For this reason, we compared the whole genome of eight isolates recovered between 2001 and 2008 at the Lausanne hospital. The genetic comparison of these isolates revealed that their genomes are extremely closely related. Yet, a few more important genetic changes, such as the replacement of a plasmid, the loss of large fragments of DNA, or the insertion of transposases, were observed. These transfers of mobile genetic elements shaped the evolution of the ST228 lineage that spread within the Lausanne hospital. Nevertheless, although the strains analyzed differed in their dynamics, we have not been able to link a particular genetic element with spreading success. Finally, the present study showed that new sequencing technologies improve considerably the quality and quantity of information obtained for a single strain; but this information is still difficult to interpret and important investments are required for the technology to become accessible for routine investigations.

Integrated Genetic Epidemiology of Infectious Diseases: The Chagas Model

Genetic typing of pathogenic agents and of vectors has known impressive developments in the last 10 years, thanks to the progresses of molecular biology, and to the contribution of the concepts of evolutionary genetics. Moreover, we know more and more on the genetic susceptibility of man to infectious diseases. I propose here to settle a new, synthetic field of research, which I call `integrated genetic epidemiology of infectious diseases' (IGEID). I aim at evaluating, by an evolutionary genetic approach, the respective impact, on the transmission and pathogenicity of infectious diseases, of the host's, the pathogen's and the vector's genetic diversity, and their possible interactions (co-evolution phenomena). Chagas' disease constitutes a fine model to develop the IGEID methodology, by both field and experimental studies.

Vancomycin-resistant Enterococci in Intensive Care Hospital Settings

Vancomycin-resistant enterococci (VRE) have recently emerged as a nosocomial pathogen and present an increasing threat to the treatment of severely ill patients in intensive-care hospital settings. We outline results of a study of the epidemiology of VRE transmission in ICUs and define a reproductive number R0; the number of secondary colonization cases induced by a single VRE-colonized patient in a VRE-free ICU, for VRE transmission. For VRE to become endemic requires R0 >1. We estimate that in the absence of infection control measures R0 lies in the range 3-4 in defined ICU settings. Once infection control measures are included R0=0.6, suggesting that admission of VRE-colonized patients can stabilize endemic VRE.

LACK-reactive CD4+ T cells require autocrine IL-2 to mediate susceptibility to Leishmania major.

Mice from most inbred strains are resistant to infection with Leishmania major whereas mice from BALB strains are highly susceptible. Resistance and susceptibility result from the development of Th1 or Th2 cells, respectively. In this report, we document an IL-2 mRNA burst, preceding the reported early IL-4 response, in draining lymph nodes of susceptible mice infected with L. major. Neutralization of IL-2 during the first days of infection redirected Th1 cell maturation and resistance to L. major, through interference with the rapid IL-4 transcription in Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) cells. A burst of IL-2 transcripts also occurred in infected C57BL/6 mice that do not mount an early IL-4 response. However, although the LACK protein induced IL-2 transcripts in susceptible mice, it failed to trigger this response in resistant C57BL/6 mice. Reconstitution experiments using C.B.-17 SCID mice and LACK-reactive CD4(+) T cells from IL-2(-/-) BALB/c mice showed that triggering of the early IL-4 response required autocrine IL-2. Thus, in C57BL/6 mice, the inability of LACK-reactive CD4(+) T cells to express early IL-4 mRNA transcription, important for disease progression, appears due to an incapacity of these cells to produce IL-2.

Interactions between learning and immunity in Drosophila melanogaster

Learning and immunity are two adaptive traits with roles in central aspects of an organism's life: learning allows adjusting behaviours in changing environments, while immunity protects the body integrity against parasites and pathogens. While we know a lot about how these two traits interact in vertebrates, the interactions between learning and immunity remain poorly explored in insects. During my PhD, I studied three possible ways in which these two traits interact in the model system Drosophila melanogaster, a model organism in the study of learning and in the study of immunity. Learning can affect the behavioural defences against parasites and pathogens through the acquisition of new aversions for contaminated food for instance. This type of learning relies on the ability to associate a food-related cue with the visceral sickness following ingestion of contaminated food. Despite its potential implication in infection prevention, the existence of pathogen avoidance learning has been rarely explored in invertebrates. In a first part of my PhD, I tested whether D. melanogaster, which feed on food enriched in microorganisms, innately avoid the orally-acquired 'novel' virulent pathogen Pseudomonas entomophila, and whether it can learn to avoid it. Although flies did not innately avoid this pathogen, they decreased their preference for contaminated food over time, suggesting the existence of a form of learning based likely on infection-induced sickness. I further found that flies may be able to learn to avoid an odorant which was previously associated with the pathogen, but this requires confirmation with additional data. If this is confirmed, this would be the first time, to my knowledge, that pathogen avoidance learning is reported in an insect. The detrimental effect of infection on cognition and more specifically on learning ability is well documented in vertebrates and in social insects. While the underlying mechanisms are described in detail in vertebrates, experimental investigations are lacking in invertebrates. In a second part of my PhD, I tested the effect of an oral infection with natural pathogens on associative learning of D. melanogaster. By contrast with previous studies in insects, I found that flies orally infected with the virulent P. entomophila learned better the association of an odorant with mechanical shock than uninfected flies. The effect seems to be specific to a gut infection, and so far I have not been able to draw conclusions on the respective contributions of the pathogen's virulence and of the flies' immune activity in this effect. Interestingly, infected flies may display an increased sensitivity to physical pain. If the learning improvement observed in infected flies was due partially to the activity of the immune system, my results would suggest the existence of physiological connections between the immune system and the nervous system. The basis of these connections would then need to be addressed. Learning and immunity are linked at the physiological level in social insects. Physiological links between traits often result from the expression of genetic links between these traits. However, in social insects, there is no evidence that learning and immunity may be involved in an evolutionary trade-off. I previously reported a positive effect of infection on learning in D. melanogaster. This might suggest that a positive genetic link could exist between learning and immunity. We tested this hypothesis with two approaches: the diallel cross design with inbred lines, and the isofemale lines design. The two approaches provided consistent results: we found no additive genetic correlation between learning and resistance to infection with the diallel cross, and no genetic correlation in flies which are not yet adapted to laboratory conditions in isofemale lines. Consistently with the literature, the two studies suggested that the positive effect of infection on learning I observed might not be reflected by a positive evolutionary link between learning and immunity. Nevertheless, the existence of complex genetic relationships between the two traits cannot be excluded. - L'apprentissage et l'immunité sont deux caractères à valeur adaptative impliqués dans des aspects centraux de la vie d'un organisme : l'apprentissage permet d'ajuster les comportements pour faire face aux changements de l'environnement, tandis que l'immunité protège l'intégrité corporelle contre les attaques des parasites et des pathogènes. Alors que les interactions entre l'apprentissage et l'immunité sont bien documentées chez les vertébrés, ces interactions ont été très peu étudiées chez les insectes. Pendant ma thèse, je me suis intéressée à trois aspects des interactions possibles entre l'apprentissage et l'immunité chez la mouche du vinaigre Drosophila melanogaster, qui est un organisme modèle dans l'étude à la fois de l'apprentissage et de l'immunité. L'apprentissage peut affecter les défenses comportementales contre les parasites et les pathogènes par l'acquisition de nouvelles aversions pour la nourriture contaminée par exemple. Ce type d'apprentissage repose sur la capacité à associer une caractéristique de la nourriture avec la maladie qui suit l'ingestion de cette nourriture. Malgré les implications potentielles pour la prévention des infections, l'évitement appris des pathogènes a été rarement étudié chez les invertébrés. Dans une première partie de ma thèse, j'ai testé si les mouches, qui se nourrissent sur des milieux enrichis en micro-organismes, évitent de façon innée un 'nouveau' pathogène virulent Pseudomonas entomophila, et si elles ont la capacité d'apprendre à l'éviter. Bien que les mouches ne montrent pas d'évitement inné pour ce pathogène, elles diminuent leur préférence pour de la nourriture contaminée dans le temps, suggérant l'existence d'une forme d'apprentissage basée vraisemblablement sur la maladie générée par l'infection. J'ai ensuite observé que les mouches semblent être capables d'apprendre à éviter une odeur qui était au préalable associée avec ce pathogène, mais cela reste à confirmer par la collecte de données supplémentaires. Si cette observation est confirmée, cela sera la première fois, à ma connaissance, que l'évitement appris des pathogènes est décrit chez un insecte. L'effet détrimental des infections sur la cognition et plus particulièrement sur les capacités d'apprentissage est bien documenté chez les vertébrés et les insectes sociaux. Alors que les mécanismes sous-jacents sont détaillés chez les vertébrés, des études expérimentales font défaut chez les insectes. Dans une seconde partie de ma thèse, j'ai mesuré les effets d'une infection orale par des pathogènes naturels sur les capacités d'apprentissage associatif de la drosophile. Contrairement aux études précédentes chez les insectes, j'ai trouvé que les mouches infectées par le pathogène virulent P. entomophila apprennent mieux à associer une odeur avec des chocs mécaniques que des mouches non infectées. Cet effet semble spécifique à l'infection orale, et jusqu'à présent je n'ai pas pu conclure sur les contributions respectives de la virulence du pathogène et de l'activité immunitaire des mouches dans cet effet. De façon intéressante, les mouches infectées pourraient montrer une plus grande réactivité à la douleur physique. Si l'amélioration de l'apprentissage observée chez les mouches infectées était due en partie à l'activité du système immunitaire, mes résultats suggéreraient l'existence de connections physiologiques entre le système immunitaire et le système nerveux. Les mécanismes de ces connections seraient à explorer. L'apprentissage et l'immunité sont liés sur un plan physiologique chez les insectes sociaux. Les liens physiologiques entre les caractères résultent souvent de l'expression de liens entre ces caractères au niveau génétique. Cependant, chez les insectes sociaux, il n'y a pas de preuve que l'apprentissage et l'immunité soient liés par un compromis évolutif. J'ai précédemment rapporté un effet positif de l'infection sur l'apprentissage chez la drosophile. Cela pourrait suggérer qu'une relation génétique positive existerait entre l'apprentissage et l'immunité. Nous avons testé cette hypothèse par deux approches : le croisement diallèle avec des lignées consanguines, et les lignées isofemelles. Les deux approches ont fournies des résultats similaires : nous n'avons pas détecté de corrélation génétique additive entre l'apprentissage et la résistance à l'infection avec le croisement diallèle, et pas de corrélation génétique chez des mouches non adaptées aux conditions de laboratoire avec les lignées isofemelles. En ligne avec la littérature, ces deux études suggèrent que l'effet positif de l'infection sur l'apprentissage que j'ai précédemment observé ne refléterait pas un lien évolutif positif entre l'apprentissage et l'immunité. Néanmoins, l'existence de relations génétiques complexes n'est pas exclue.

Probiotic microorganisms: 100 years of innovation and efficacy. Modes of action

AbstractArticle StructureFigures and TablesReferences Benefits from probiotic micro-organisms have been recognised for over 100 years, and as being useful in poultry for 50 years. Fuller (1989) redefined probiotics as ‘a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance’. Benefits derived from this improved intestinal microbial balance could be reflected in performance or prevention of pathogen colonisation. Probiotic micro-organisms use in poultry production has been widely accepted and new opportunities arose from the 2006 EU ban on antimicrobial growth promoters. The majority of microbial products for compound feeds are made up from a relatively small number of micro-organisms that are normally present in the GI tract. They include non-sporulated bacteria, sporulated bacteria, fungi or yeasts; and presented from single to multi-strain products. A review on the proposed modes of action is presented including recent approaches to quorum sensing interference

Human Plasma-derived Polymeric IgA and IgM Antibodies Associate with Secretory Component to Yield Biologically Active Secretory-like Antibodies.

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ∼15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins.

A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria.

Bacteria can be refractory to antibiotics due to a sub-population of dormant cells, called persisters that are highly tolerant to antibiotic exposure. The low frequency and transience of the antibiotic tolerant "persister" trait has complicated elucidation of the mechanism that controls antibiotic tolerance. In this study, we show that 2' Amino-acetophenone (2-AA), a poorly studied but diagnostically important small, volatile molecule produced by the recalcitrant gram-negative human pathogen Pseudomonas aeruginosa, promotes antibiotic tolerance in response to quorum-sensing (QS) signaling. Our results show that 2-AA mediated persister cell accumulation occurs via alteration of the expression of genes involved in the translational capacity of the cell, including almost all ribosomal protein genes and other translation-related factors. That 2-AA promotes persisters formation also in other emerging multi-drug resistant pathogens, including the non 2-AA producer Acinetobacter baumannii implies that 2-AA may play an important role in the ability of gram-negative bacteria to tolerate antibiotic treatments in polymicrobial infections. Given that the synthesis, excretion and uptake of QS small molecules is a common hallmark of prokaryotes, together with the fact that the translational machinery is highly conserved, we posit that modulation of the translational capacity of the cell via QS molecules, may be a general, widely distributed mechanism that promotes antibiotic tolerance among prokaryotes.

Lassa virus entry in antigen presenting cells and evaluation of a novel nanoparticle vaccine platform against arena viruses

Arenaviruses are a large and diverse family of viruses that merit significant attention as causative agents of severe hemorrhagic fevers in humans. Lassa virus (LASV) in Africa and the South American hemorrhagic fever viruses Junin (JUNV), Machupo (MACV), and Guanarito (GTOV) have emerged as important human pathogens and represent serious public health problems in their respective endemic areas. A hallmark of fatal arenaviruses hemorrhagic fevers is a marked immunosuppression of the infected patients. Antigen presenting cells (APCs) such as macrophages and in particular dendritic cells (DCs) are early and preferred targets of arenaviruses infection. Instead of being recognized and presented as foreign antigens by DCs, arenaviruses subvert the normal mechanisms of pathogen recognition, invade DCs and establish a productive infection. Viral replication perturbs the DCs' ability to present antigens and to activate T and B cells, contributing to the marked virus-induced immunosuppression observed in fatal disease. Considering their crucial role in the development of an anti-viral immune response, the mechanisms by which arenaviruses, and in particular LASV, invade DCs are of particular interest. The C-type lectin DC-specific Intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) was recently identified as a potential entry receptor for LASV. The first project of my thesis focused therefore on the investigation of the role of DC-SIGN in LASV entry into primary human DCs. My data revealed that DC-SIGN serves as an attachment factor for LASV on human DCs and can facilitate capture of free virus and subsequent cell entry. However, in contrast to other emerging viruses, of the phlebovirus family, I found that DC-SIGN does likely not function as an authentic entry receptor for LASV. Moreover, I was able to show that LASV enters DCs via an unusually slow pathway that depends on actin, but is independent of clathrin and dynamin. Considering the lack of effective treatments and the limited public health infrastructure in endemic regions, the development of protective vaccines against arenaviruses is an urgent need. To address this issue, the second project of my thesis aimed at the development of a novel recombinant arenavirus vaccine based on a nanoparticle (NPs) platform and its evaluation in a small animal model. During the first phase of the project I designed, produced, and characterized suitable vaccine antigens. In the second phase of the project, I generated antigen-conjugated NPs, developed vaccine formulations, and tested the NPs for their ability to elicit anti-viral T cell responses as well as anti-viral antibodies. I demonstrated that the NPs platform is able to activate both cellular and humoral branches of the adaptive anti-viral immunity, providing proof-of-principle. In sum, my first project will allow, in a long term perspective, a better understanding of the viral pathogenesis and contribute to the development of novel antiviral strategies. The second project will expectidly offer a new treatment option against arenaviruses.

Discovery of new intracellular pathogens by amoebal coculture and amoebal enrichment approaches.

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

A review of the ticks (Acari, Ixodida) of Brazil, their hosts and geographic distribution - 1. The State of Rio Grande do Sul, Southern Brazil

A review of the ticks (Acari, Ixodida) of the State of Rio Grande do Sul, southern Brazil, was completed as a step towards a definitive list (currently indicated as 12) of such species, their hosts and distribution. The ticks: Argas miniatus (poultry), Ixodes loricatus (opossums), Amblyomma aureolatum (dogs), A. calcaratum (anteaters), A. cooperi (capybaras), A. nodosum (anteaters), A. tigrinum (dogs) (Neotropical) and Rhipicephalus sanguineus (dogs) (introduced, cosmopolitan, Afrotropical) were confirmed as present, in addition to the predominant, Boophilus microplus (cattle) (introduced, pan-tropical, Oriental). Of the further 18 species thus far reported in the literature as present in the state, but unavailable for examination: only Ornithodoros brasiliensis (humans and their habitations) (Neotropical), Ixodes affinis (deer) (Nearctic/Neotropical) and I. auritulus (birds) (Nearctic/Neotropical/Afrotropical/ Australasian) are considered likely; 13 species would benefit from corroborative local data but the majority appear unlikely; reports of A. maculatum (Nearctic/Neotropical, but circum-Caribbean) are considered erroneous; the validity of A. fuscum is in doubt. The very recent, first known report of the tropical Anocentor nitens (horses)(Nearctic/Neotropical), but still apparent absence of the tropical A. cajennense (catholic) (Nearctic/Neotropical) and the sub-tropical/temperate Ixodes pararicinus (cattle) (Neotropical) in Rio Grande do Sul are important for considerations on their current biogeographical distribution and its dynamics in South America. The state has relatively long established, introduced ("exotic"), Old World tick species (B. microplus, R. sanguineus) that continue to represent significant pests and disease vectors to their traditional, introduced domestic animal hosts, cattle and urban dogs. There are also indigenous, New World ticks (A. miniatus, O. brasiliensis, A. aureolatum, A. nitens), as both long established and possibly newly locally introduced species in the state, that should be considered as potential and emergent pests and pathogen vectors to humans and their more recently acquired, introduced domestic animal hosts; rural poultry, dogs and horses.