The aim of this study was to develop improved methods to detect viable Coxiella burnetii in wastes from livestock production. The impetus for this work arose because there is a significant risk of infection for humans attributed to contact with waste products from the livestock production industry. This situation is further compounded by the lack of suitable tools to detect viable C. burnetii in these wastes. In addition, effective disinfection strategies for livestock wastes are also required to reduce the risk of infection with C. burnetii for individuals that come into contact with these waste products. A quantitative real-time PCR system (qPCR) with high sensitivity and specificity was developed to detect the C. burnetii in environmental samples associated with domestic ruminants and native Australian marsupials. Different detection chemistries and procedures were evaluated based on their sensitivity, specificity and reproducibility. Overall it was found that the TaqMan PCR targeting the IS1111a locus provided the most sensitive and reproducible test. The Geneworks PowerSoil(tm) DNA isolation kit provided the best compromise between reproducibility and recovery of DNA from livestock wastes. When combined, the IS1111a TaqMan qPCR and Geneworks PowerSoil DNA Extraction Kit provided a test which was capable of detecting as few as two C. burnetii genome equivalents in 0.2g of soil or faeces. Coxiella burnetii has been shown to display extreme resistance to environmental exposure. Therefore, assessment of the viability of the organism in environmental matrices is more useful for risk assessment programs than detection of DNA alone. A quantitative reverse transcriptase PCR was developed that was able to detect viable C. burnetii cells in soil. The sensitivity of the assay was enhanced by heat-treating the soil samples prior to extraction of RNA. The factor most often associated with transfer of C. burnetii to humans is exposure to livestock or their waste. Therefore, decontamination of waste from livestock production industries is a key factor in preventing outbreaks of Q fever. A system was developed to determine the efficacy of various disinfectant treatments against the environmental pathogen C. burnetii. Treatments evaluated included sodium hypochlorite, ozone, ultraviolet light, peracetic acid (PAA), and Virkon S®. Sodium hypochlorite at a concentration of 0.1 mM reduced the infectivity of C. burnetii by over 92% while treatment with the same sodium hypochlorite concentration in wastewater showed significantly reduced efficacy. Despite this reduced potency, sodium hypochlorite is still useful for control of C. burnetii in the liquid waste of animal production. Commercially available ELISA and CFT assays exist for ruminants but there are no immunological tests available for detecting C. burnetii in marsupials even though Australian marsupials are known to be susceptible to C. burnetii. An indirect ELISA for detecting anti-Coxiella antibodies in kangaroos was developed. Paired serum and faecal samples were taken from 379 ruminants from Western Australia and the serum was tested with a commercially available ELISA and the complement fixation test while the faeces was tested using the qPCR developed during this study. Paired serum and faecal samples were taken from 343 kangaroos from WA and were tested with the antibody-ELISA developed during this study and by qPCR. A very low prevalence of anti-Coxiella antibodies was observed in the ruminants sampled and results from immunological tests correlated poorly with qPCR data. The development of an ELISA for use with kangaroo serum was problematic because of the lack of reference sera from animals known to be infected with C. burnetii. Despite this results from the ELISA developed suggested that the apparent seroprevalence in the WA animals surveyed was approximately 34%. Results from testing kangaroo faeces with the qPCR correlated poorly with the results from the antibody-ELISA. These data suggest that kangaroos may be a significant reservoir of C. burnetii in Western Australia and due to cohabitation of kangaroos and domestic ruminants, may provide a link between the wildlife and domestic cycles of C. burnetii.

Coxiella burnetii is an intracellular pathogen that can cause both acute and chronic disease (Q fever) in humans and infects many animals with varying clinical illness and persistence. A guinea pig aerosol-challenge model of acute Q fever was developed using infection with C. burnetii across a 5-log range of challenge doses. Clinical signs included fever, weight loss, respiratory difficulty, and death, with degree and duration of response corresponding to dose of organism delivered. Histopathologic evaluation revealed coalescing panleukocytic bronchointerstitial pneumonia 7 days after a high-dose challenge, resolving to multifocal lymphohistiocytic interstitial pneumonia by 28 days. Clinical and pathologic changes noted in these guinea pigs were comparable to those seen in human acute Q fever, making this an accurate and valuable animal model. This model was used to compare the relative virulence of eight isolates from four different genotypic groups: I (RSA493, RSA334, and RSA270), IV (Q177 and Q173), V (Q212 and Q217), and VI (5J108-111). Guinea pigs infected with group I acute-diseaseassociated isolates had severe respiratory disease, while no to moderate clinical illness was observed in animals given group IV or V chronic-disease-associated isolates. 5J108- 111 appeared avirulent. These data suggest that C. burnetii isolates have a range of disease potentials and support a distinction in strain virulence between established genotypic groups, though isolates within the same genomic group cause similar pathologic responses. Heterologous protection was confirmed by cross vaccination and challenge with RSA493 and Q217. A marked non-specific suppression of lymphoproliferation was noted at 14 and 28 days post infection with RSA493; similar suppression was seen after infection with Q173 and Q212 but not 5J108-111. Proinflammatory cytokines IFN-γ and TNF-α were produced during early C. burnetii infection, at which time anti-inflammatory cytokines TGF-β and IL-10 were repressed. A vaccine made from phase I C. burnetii was found to be completely protective against lethal infection in the guinea pig model, while vaccination with killed phase II organisms conferred only partial protection, preventing death and reducing but not precluding fever and respiratory illness. Protective vaccination significantly stimulated cell-mediated immunity and elicited increases in IFN-γ, TNF-α, and IL-12p40 mRNA levels.

Thesis (Ph.D.) -- University of Montana, 2008. / Title from author supplied metadata. Description based on contents viewed on June 26, 2009. Includes bibliographical references.

Made available in DSpace on 2015-10-23T12:47:04Z (GMT). No. of bitstreams: 2 ana_silva_ioc_mest_2014.pdf: 1478235 bytes, checksum: 6d52627a1a269a7288d87d75c2559e6c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-04-14 / Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, / O aumento do consumo de drogas ilícitas na atualidade vem acompanhado não apenas do impacto social e econômico, como também na saúde. As pessoas que injetam drogas apresentam maior susceptibilidade a infecções por diversos agentes, devido ao uso da droga e à subsequente imunossupressão, como também pelo risco de maior exposição a agentes transmitidos por artrópodes e reservatórios vertebrados em ambientes insalubres. São muitos os estudos correlacionando o consumo de drogas à infecção pelo HIV, VHB e VHC, porém raros investigam outros agentes. No presente estudo transversal e retrospectivo, selecionamos aleatoriamente 300 amostras de um estudo multicêntrico da década de 1990 e fizemos a pesquisa sorológica para Bartonella spp., Coxiella burnetii e Hantavirus. O perfil demográfico e social encontrado mostrou-se em concordância com a literatura: homens, jovens, de baixa renda e com histórico de prisão. A maior parte apresentava situações de risco por injetar drogas com dispositivos previamente usados e por injetar em local público. Alguns fatores foram discordantes da literatura internacional e podem ter influenciado os resultados, uma vez que são considerados situações de risco, como escassez de pessoas que injetaram drogas enquanto presas, que residem em instituições, ou sem-teto A evidência de infecção por C. burnetii foi de 9,3%, praticamente o dobro da observada na população geral brasileira, embora, em comparação com dados internacionais, a prevalência tenha sido menor. Não houve diferença estatística significativa em relação às variáveis demográficas entre os grupos sororeativos e soronegativos para febre Q. A presença de anticorpos anti-Bartonella foi de 1%, bem discrepante dos estudos nacionais e internacionais. Pode ser justificado pela não inclusão no teste sorológico de antígeno de B. elizabethae e de B. quintana e pela reduzida frequência de sem-tetos. Não houve correlação do alto consumo de álcool com maior soroprevalência por Bartonella spp., provavelmente pelos motivos acima citados. A soroprevalência para hantavirose de 4% foi semelhante à descrita em estudo brasileiro na população geral. Em relação aos usuários de drogas injetáveis, somente um artigo americano evidenciou uma soroprevalência bem menor, de 0,2%. Isso indica que em meios urbanos brasileiros, hantavírus, provavelmente o Seoul, pode estar circulando. Embora considerando que o estudo foi retrospectivo com base em evidência sorológica e que a possibilidade de reação cruzada não pode ser totalmente descartada, o presente estudo aponta para a importância de se incluir a pesquisa de outros agentes infecciosos além do HIV e dos vírus das hepatites B e C na população injetora de drogas / The increased consumption of illicit drugs today is followed not only by a social and economic impact, as well as in health. People who inject drugs are more susceptible to infections by various agents due to drug use and subsequent immunosuppressi on, as well as the risk of increased exposure to agents transmitted by arthropods and vertebrate reservoirs in unhealthy environments. Many studies link drug use to HIV, HBV and HCV, however only a few investigate other agents. In this re trospective cross - sectional study, we selected randomly 300 samples from a multicenter study from the 1990s and made a serological survey for Bartonella spp., Coxiella burnetii and hantavirus. The demographic and social profile was found in agreement with the literature: me n, young age , low income and with a history of imprisonment . The majority was exposed to risk situations by injecting drugs with previously used devices and by injecting in a public place s . Some factors were dis agreeing with the international literature an d may have influenced the results, since they are considered high - risk situations, such as shortage of people who injected drugs while imprisoned, residing in institutions, or homeless. Evidence of C. burnetii infection was 9.3%, almost double the rate in the general Brazilian population, although in comparison with international data, the prevalence was lower. There was no statistically significant difference in relation to demographic variables among seroreactive and seronegative groups for fever Q. The p resence of antibodies to Bartonella was 1%, significantly discrepant from national/ international studies. Possibly justified by the non inclusion of antigen B. elizabethae and B. quintana in the serological test , and the reduced frequency of homelessness. There was no correlation between high alcohol consumption with higher seroprevalence of Bartonella spp., p robably for the reasons mentioned above. The seroprevalence of hantavirus 4% was similar to that described in a Brazilian study in the general populat ion. Regarding inje c t ing drug users , only an American paper showed a much lower prevalence of 0.2%. This indicates that in Brazilian urban areas, hanta virus can circulate , probably Seoul hantavirus . While considering that the study was retrospective based on serological evidence and the possibility of cross - reactivity cannot be completely ruled out, this study points to the importance of including the research of other infectious agents other than H IV and hepatitis viruses B and C in injectin g drug population

Made available in DSpace on 2016-04-07T13:25:46Z (GMT). No. of bitstreams: 2 maria_guia_ioc_dout_2015.pdf: 33668716 bytes, checksum: 116b84e24b72cbb6e3a9a67cb4657da1 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Febre Q é uma zoonose cosmopolita causada por Coxiella burnetii, uma bactéria intracelular obrigatória gram-negativa da ordem Legionellales. A doença, que ocorre como pequenos surtos ou casos isolados, tem amplo espectro clínico, desde doença febril limitada, pneumonia, hepatite à endocardite e meningoencefalite. Pequenos roedores silvestres são importantes reservatórios, mas a infecção humana está principalmente relacionada a ovelhas, cabras e gado bovino, embora gatos, cães e coelhos estejam implicados em surtos urbanos. Na população humana a transmissão ocorre por aerossóis provenientes de líquido amniótico, placenta e lã, além da urina, fezes, leite e outras secreções animais contendo o agente. No Brasil, a primeira descrição de febre Q ocorreu em 1953. Embora casos esporádicos confirmados por teste sorológico e estudos sorológicos tenham apontado para a presença de C. burnetii no Brasil, somente em 2008 o agente foi identificado por análise molecular em paciente no município de Itaboraí/RJ. Com a consequente caracterização da infecção também nos animais domésticos de propriedade do paciente, durante o período de 2010-2011, o presente estudo foi proposto com o objetivo de: (i) realizar a vigilância de febre Q em pacientes com suspeita de dengue internados no Hospital Municipal Desembargador Leal Júnior, em Itaboraí, durante o período de 24 meses; (ii) verificar a presença de infecção nos familiares dos casos de febre Q; (iii) analisar os animais de propriedade dos casos de febre Q; (iv) investigar a presença de C. burnetii em animais silvestres capturados nas áreas de onde procederam os casos de febre Q e (v) pesquisar C. burnetii nos artrópodes coletados nos animais Neste estudo foram utilizados o teste de imunofluorescência indireta e a análise molecular (PCR) visando os elementos IS1111 transposase no genoma de C. burnetii. Dos 272 pacientes atendidos no período de 2013 a 2014, 26 (10%) apresentaram anticorpos anti-C. burnetii e nove (3,3%) foram PCR positivos. Um dos pacientes apresentou também infecção por dengue. A análise das sequências genômicas obtidas demonstrou a elevada similaridade entre si (99-100%) e com as sequências de C. burnetii depositadas no GenBank. Dos 35 animais domésticos estudados, seis foram sororreativos: 1/13 cães, 3/13 gatos, 2/9 ovelhas. A análise molecular foi positiva em swab anal de um filhote de gato e em amostra de tecido do úbere da ovelha sororreativa com história de aborto. Todos os 59 animais silvestres dos gêneros Didelphis, Philander, Micoureus, Akodon, Oligoryzomys e Nectomys foram negativos na análise molecular. Dos 283 artrópodes analisados, DNA de C. burnetii foi amplificado em oito dos 266 exemplares de Rhipicephalus sanguineus e em um exemplar de oito Amblyomma sculptum e nenhum dos sete Dermacentor nitens e duas pulgas Ctenocephalides canis identificados. Os resultados comprovam a presença de C burnetii em Itaboraí, confirmam a necessidade da inclusão da febre Q no diagnóstico diferencial da dengue e alertam para a necessidade da sensibilização dos profissionais de saúde sobre a ocorrência desta zoonose no Brasil / Q fever is a worldwide zoonosis disease caused by Coxiella burnetii, a gram-negative obligate intracellular bacterium of the legionellales order. The disease, which occurs as individual cases or small outbreaks have broad-spectrum clinical manifestation from limited febrile disease, pneumonia, endocarditis, hepatitis and meningoencephalitis. Small wild rodents are important reservoirs, but human infection is mainly related to sheep, goats and cattle, although cats, dogs and rabbits are involved in urban outbreaks. In human population, transmission occurs by aerosol from amniotic fluid, placenta and wool, as well as urine, feces, milk and other animal secretions, containing the agent. In Brazil, the first Q fever description occurred in 1953. Although sporadic cases confirmed by serological testing and sero-epidemiological studies have pointed to the presence of C. burnetii in Brazil, only in 2008 it was possible identify the agent in a patient's municipality of Itaboraí/RJ by molecular assay. With the consequent characterization of the infection also in domestic animals owned by the patient during the period 2010-2011, this study was proposed with the aim of: (i) conduct surveillance of Q fever in hospitalized patients with suspected dengue in Itaboraí at the Hospital Municipal Desembargador Leal Junior, during the period of 24 months; (ii) to verify the presence of Q fever infection in family cases ; (iii) analyzing, animals the property Q fever cases; (iv) investigate the presence of C. burnetii in wild animals captured in areas from which proceeded cases of Q fever and (v) search C burnetii in arthropods collected from animals. This study used the indirect immunofluorescence assay and molecular analysis (PCR) targeting the IS1111 transposase elements in the genome of C. burnetii. Of the 272 patients assisted from 2013 to 2014, 26 (10%) had anti-C. burnetii antibodies, and nine (3.3%) were PCR positive. One of these patients had also dengue infection. The analysis of the genomic sequences obtained showed high similarity to each other (99-100%) and the sequences of C. burnetii in GenBank. Of the 35 domestic animals studied, six were seroreactive: 1/13 dogs, cats 3/13, 2/9 sheep. Molecular analysis was positive in anal swab of a young kitten and tissue sample from the udder of sororreativa sheep with abortion history. All 59 wild animals Didelphis, Philander, Micoureus, Akodon, Oligoryzomys and Nectomys were negative by molecular analysis. Of the 283 arthropods analyzed, C. burnetii DNA was amplified in eight of 266 Rhipicephalus sanguineus and a specimen of 8 Amblyomma sculptum and none of 7 Dermacentor nitens and 2 fleas Ctenocephalides canis identified. The results show the presence of C. burnetii in Itaboraí, confirm the need for inclusion of Q fever in dengue differential diagnosis and point to the need of awareness among health professionals about the occurrence of this zoonosis in Brazil.

Indiana University-Purdue University Indianapolis (IUPUI) / Coxiella burnetii, the zoonotic agent of human Q fever and chronic endocarditis, is an obligate intracellular bacterial pathogen. The Coxiella intracellular niche, a large, lysosome-like parasitophorous vacuole (PV), is essential for bacterial survival and replication. There is growing evidence that host cell cholesterol trafficking plays a critical role in PV development and maintenance, prompting an examination of the role of cholesterol-binding host protein ORP1L (Oxysterol binding protein-Related Protein 1, Long) during infection. ORP1L is a multi-functional cholesterol-binding protein involved in late endosome/lysosome (LEL) trafficking, formation of membrane contact sites between LEL and the endoplasmic reticulum (ER), and cholesterol transfer from LEL to the ER. ORP1L localizes to the PV at novel membrane contact sites between the ER and the PV membrane. Ectopically expressed ORP1L in Coxiella-infected cells localizes to the PV membrane early during infection, before significant PV expansion and independent of other PV-localized proteins. Further, the N-terminal ORP1L Ankyrin repeats are both necessary and sufficient for PV localization, suggesting that protein-protein interactions, and not protein-lipid interactions, are primarily involved in PV association. Coxiella employs a Type IVB Secretion System (T4BSS) to translocate effector proteins into the host cytoplasm and manipulate various cellular functions. ORP1L is not found on the PV of a Coxiella mutant lacking a functional T4BSS, indicating a secreted bacterial protein is likely responsible for ORP1L recruitment. We identified a Coxiella mutant with a transposon insertion in CBU_0352 that exhibits a 50% decrease in ORP1L recruitment, suggesting that Coxiella CBU_0352 interacts directly or indirectly with ORP1L. Finally, we found that ORP1L depletion using siRNA alters PV dynamics, resulting in smaller yet more fusogenic Coxiella PVs. Together, these data suggest that ORP1L is specifically recruited to the PV, where it plays a novel role in Coxiella PV development and interactions between the PV and the host cell.

Parmi les arthropodes, les tiques sont les plus importants vecteurs de pathogènes en termes de diversité et sont la première cause de transmission de maladies vectorielles en Europe et Amérique du Nord. Si ces pathogènes font l'objet de nombreux travaux, les tiques hébergent aussi d'autres symbiotes qui contribuent de manière importante à leur phénotype. Au cours des dernières années, de nombreuses bactéries symbiotiques ont ainsi été recensées chez les tiques. Parmi celles-ci des bactéries présentant une forte homologie avec Coxiella burnetii ont été découvertes. Contrairement à C. burnetii, l'agent responsable de la fièvre Q, les Coxiella-like endosymbiotiques (Coxiella-LE) ne semblent pas capables d'infecter d'autres hôtes que les tiques. Elles font partie des symbiotes à transmission maternelle les plus répandus chez les espèces de tiques et pourraient jouer un rôle important dans la biologie de ces arthropodes. Des premiers éléments suggèrent en effet, que les Coxiella-LE pourraient avoir un rôle nutritionnel en synthétisant vitamines B et cofacteurs absents de l'alimentation de leur hôte tique. Pour comprendre les relations entretenues entre les Coxiella-LE et leurs hôtes je me suis intéressé,au cours de mes travaux de thèse, a l'évolution du genre Coxiella. Pour cela des approches d'analyses phylogénétiques et de génomique comparative ont été utilisées. J'ai ainsi participe à l'établissement de la phylogénie du genre Coxiella par Multi-Locus Sequence Typing (MLST), qui a permis de mettre en évidence la diversité de ce genre bactérien. De manière intéressante C. burnetii émerge au sein d'un de ces clades de bactéries endosymbiotiques de tiques, ce qui semble témoigner d'une récente transition vers la pathogénie. Nous avons séquencé deux nouveaux génomes de Coxiella-LE afin de réaliser une étude de génomique comparative. Tous les génomes de Coxiella étudiés, y compris ceux de C. burnetii, possèdent les gènes nécessaires à la biosynthèse des vitamines B et des cofacteurs, retrouvés habituellement chez les symbiotes nutritionnels d'arthropodes hématophages. Cette découverte renforce l'idée d'un rôle important des Coxiella-LE pour leur hôte tique et, d'après la phylogénie, l'ancêtre commun de ces bactéries serait donc un endosymbiote mutualiste de tique. Pourtant des traces de gènes impliqués dans la virulence de Coxiella burnetii ont été retrouvées dans des génomes appartenant à des clades distincts de Coxiella-LE, ce qui semble plutôt indiquer des pertes récurrentes de la virulence. De plus, différents niveaux d'érosion génomique sont retrouvés dans les génomes de Coxiella-LE étudiés, ce qui indiquerait de fréquents transferts d'hôtes. De tels transferts expliqueraient l'absence de co-cladogenese entre la phylogénie des Coxiella-LE et celle de leur hôte, une caractéristique originale pour un symbiote qui semble obligatoire. Par ailleurs, plusieurs symbiotes à transmission maternelle peuvent être retrouvés chez les tiques, le deuxième axe de ma thèse s'intéresse à l'impact de ces co-infections. Pour cela une population de tiques appartenant à l'espèce Dermacentor marginatus a été étudiée. Cette espèce est, en effet, fréquemment infectée par des bactéries Coxiella-LE, Rickettsia et Spiroplasma et différents statuts d'infection peuvent être observés chez les individus. Aucune compétition n'a été démontrée entre ces bactéries, puisqu’aucune n'interfère avec la transmission et la densité des autres. Néanmoins, en cas de triple infection, la valeur adaptative des hôtes est fortement diminuée avec une réduction importante de leur taille (10%). La transmission verticale de ces symbiotes n'étant pas complète, il devient alors difficile de comprendre comment ces bactéries atteignent de si fortes prévalences au sein de la population avec des coûts associés aussi importants [etc…] / Among arthropods, ticks are the most important vectors of pathogens in terms of diversity and are the leading cause of transmission of vector-borne diseases in Europe and North America. While these pathogens are the most studied, ticks also harbor other symbionts that contribute significantly to their phenotype. Recently many symbiotic bacteria have been described in ticks. Among them, bacteria exhibiting strong homology with Coxiella burnetii have been discovered. Unlike C. burnetii, the causative agent of Q fever, Coxiella-Like Endosymbiont (Coxiella-LE) seems unable to infect other hosts than ticks. They are among the most widespread maternally-inherited symbionts in tick species and could play an important role in their biology. Coxiella-LE may indeed have a nutritional role by synthesizing B vitamins and cofactors absent from their host's diet. To understand the interaction between Coxiella-LE and their hosts, my thesis work focused on the evolution of the Coxiella genus. For this purpose, phylogenetic analyzes and comparative genomic approaches have been carried out. I have participated in the establishment of the phylogeny of the Coxiella genus by Multi-Locus Sequence Typing (MLST), which highlights the diversity of this bacterial genus. Interestingly C. burnetii emerges within one of these clads of tick endosymbiotic bacteria, which may suggest a recent transition towards pathogenicity. Two new genomes of Coxiella-LE were sequenced to perform comparative genomic analyses. All Coxiella genomes studied, including those of C. burnetii, possess the genes encoding for the biosynthesis of B vitamins and cofactors, as usually found in nutritional symbionts of blood-sucking arthropods. This result strengthens the idea of an important role of Coxiella-LE for their host ticks and, according to the phylogeny, the common ancestor of these bacteria was therefore a mutualistic tick endosymbiont. However, traces of genes involved in the virulence of C. burnetii have been found in genomes belonging to distinct clads of Coxiella-LE, which rather indicate recurrent losses of virulence. Moreover, different levels of genomic erosion are found in the genomes of Coxiella-LE studied, which could indicate different transitions towards the mutualistic way of life. Such recurrent transfers would explain the absence of cocladogenesis between Coxiella-LE and their host phylogeny, an uncommon feature for an obligatory symbiont. As several maternally-inherited symbionts can be found in ticks, the second axis of my thesis has focused on the impact of co-infections. For this purpose, a population of ticks belonging to the species Dermacentor marginatus was studied. This species is frequently infected with Coxiella-LE, Rickettsia and Spiroplasma bacteria and different infection status can be observed in individuals from a single population. No competition has been demonstrated between these bacteria, since none interferes with the transmission and density of the others. However, in case of triple infection, the fitness of the host appears greatly reduced with a significant reduction in size (10%). Since vertical transmission of these symbionts is incomplete, understanding how these symbionts and co-infections are maintained despite this significant cost remains an open question. If the symbiotic strategies of these symbionts are still unknown, it is likely that their transmission is not only maternal, but also horizontal [etc…]

Coxiella burnetii est l’agent pathogène responsable de la fièvre Q. Dans le cadre de cette thèse nous nous sommes intéressés à l'étude de souches de C.burnetii responsables d'événements épidémiques. Nous avons séquencé une souche de génotype MST33 (Z3055), proche de la souche responsable de l'épidémie de fièvre Q aux Pays-Bas, et une souche de génotype MST17 (Cb175) clone provoquant l'une des formes les plus virulentes de fièvre Q aiguë jamais décrite auparavant et retrouvée à ce jour uniquement en Guyane Française. Les résultats de ces analyses montrent que le génome de la souche Z3055 était très similaire à celui de la souche de référence Nine Mile I. Les différences observées sont liées à la présence de mutations non synonymes dans le génome de Z3055. Le pourcentage élevé de protéines membranaires mutées pourrait expliquer l’ampleur de cette épidémie en Hollande. En effet, le changement de profil antigénique pourrait être à l’origine de la formation d’un nouveau sérotype capable d'échapper à la réponse immunitaire de l'hôte et de diffuser facilement dans une population au système immunitaire naïf. Nous avons d’ailleurs montré que la souche responsable de la fièvre Q en Guyane (Cb175) présente des différences chromosomiques importantes par rapport à NMI. Ces différences se manifestent principalement par la présence d’une délétion d’une région de 6105pb contenant l’opéron hlyCABD du système de sécrétion de type 1 (T1SS). Ce résultat est cohérent avec ce qui a été observé chez les bactéries épidémiques les plus dangereuses comparées à leurs espèces non-épidémiques plus proches qui ont un génome réduit et contiennent moins de protéines du système de sécrétion. / Coxiella burnetii is a human pathogen that causes the zoonotic disease Q fever. In this work, we focused on the study of strains responsible for epidemic events. Particularly, we sequenced the clone of the strain responsible for Netherlands outbreak having genotype MST33 (Z3055), and strain having MST17 (Cb175) responsible for one of the most severe form of acute Q fever never reported in literature and uniquely described in French Guiana. Our findings showed that the Netherlands outbreak responsible strain (clone Z3055) was highly similar to the reference strain Nine Mile I. Only slight differences were observed, which were related to non-synonymous mutations in Z3055 genome. The high proportion of mutated membrane proteins could explain this large-scale outbreak. Change of antigenic profile may have led to a new serotype, conferring to the novel clone the capacity to escape the host immune response and to disseminate easily in a immunologically naïve population. On the contrary, the type strain responsible for Q fever in Guiana (Cb175) showed an important difference in its chromosome sequence compared to the reference NMI because of the deletion of a sequence of 6105bp containing the Type 1 secretion systems (T1SS) hlyCABD operon. This result appear consistent with previous findings that showed the most dangerous epidemic bacteria compared with their closest non-epidemic species are characterized by reduced genomes accompanied by significant decrease in ORF content and contain less secretion system proteins.

Endocardite infecciosa é uma doença associada à elevada morbidade e letalidade. O diagnóstico precoce e o reconhecimento de sua etiologia podem contribuir para o sucesso do tratamento antibiótico; entretanto, cerca de um quarto das endocardites permanece sem diagnóstico etiológico. Este estudo teve como objetivo principal identificar a frequência de endocardite por Bartonella spp. e Coxiella burnetii dentre as endocardites com culturas negativas comunitárias e avaliar os fatores preditores dessas infecções. Como objetivo secundário compararam-se as características clínicolaboratoriais e prognósticas entre as endocardites comunitárias com culturas negativas e positivas. Foram avaliados também os fatores associados à letalidade intra-hospitalar das endocardites com culturas negativas. Entre janeiro de 2004 e janeiro de 2009, foram investigados 369 episódios consecutivos de endocardite em pacientes atendidos no Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo - InCor HC-FMUSP. Foram estudados os casos que ocorreram em adultos, classificados pelos critérios de Duke modificados como \"endocardite definida\" e de origem comunitária. Assim, foram incluídos 221 episódios de endocardite, 170 com culturas positivas e 51 com culturas negativas. Neste último grupo, foram feitas as pesquisas sorológicas (reação de imunofluorescência indireta) e histopatológica de Bartonella spp. e Coxiella burnetii. Consideraram-se positivos títulos de imunoglobulina G (IgG) >= 800 para Bartonella henselae e ou Bartonella quintana, e IgG antifase I para C. burnetii > 800. O estudo histopatológico das valvas cardíacas foi capaz de identificar morfologicamente a etiologia de 87% das endocardites com culturas negativas, enquanto que o método de Gram do tecido a fresco o fez em somente 10% dos casos. As endocardites com culturas negativas apresentaram maior frequência de dispneia à admissão (p=0,001), menor valor de proteína C reativa (p=0,009), menor Fração de Ejeção do Ventrículo Esquerdo (Feve) (p=0,022) e necessitaram de mais tempo para o início do tratamento antibiótico para endocardite (p < 0,001) quando comparadas àquelas com culturas positivas. Não houve diferença estatisticamente significante entre os grupos na letalidade intra-hospitalar e na sobrevida após alta hospitalar. Verificou-se que a presença de diabetes mellitus (p=0,009) ou sepse grave na admissão (p=0,01) esteve independentemente associada ao óbito intra-hospitalar entre as endocardites com culturas negativas. Dez casos de endocardite por Bartonella spp. (frequência 19,6% [IC95%: 9,8 - 33,1]) e quatro casos de endocardite por Coxiella burnetii (frequência 7,8% [IC95%: 2,2 - 18,9]) foram diagnosticados dentre os 51 episódios de endocardite com culturas negativas. As endocardites por Bartonella spp. apresentavam menor Feve (p=0,025), associação com a identificação de cocobacilo Gram-negativo no exame histológico da valva cardíaca (p=0,001) e presença de gato no domicílio (p=0,001). Conclusões: Bartonella spp. e Coxiella burnetii foram as etiologias de quase um terço (27,5%) das endocardites comunitárias com culturas negativas. A presença de gato no domicílio, Feve <= 45%, e a identificação de cocobacilo Gramnegativo no exame histológico da valva cardíaca em pacientes com endocardite com culturas negativas parecem estar associadas à infecção por Bartonella spp. O exame histológico da valva cardíaca permitiu a identificação morfológica do micro-organismo na maioria dos casos, mesmo quando as hemoculturas estavam negativas. Não se observou diferença na letalidade intra-hospitalar e na sobrevida em longo prazo entre os dois grupos. A presença de diabetes mellitus ou sepse grave à admissão associou-se ao óbito hospitalar nas endocardites com culturas negativas / Infective endocarditis is associated with high morbidity and lethality. Early diagnosis and recognition of the specific etiology can contribute to successful antibiotic treatment. However, approximately one-fourth of endocarditis cases remain without an etiologic diagnosis. This study aimed to identify the frequency of endocarditis caused by Bartonella spp. and Coxiella burnetii among cases of community-acquired culture-negative endocarditis and to also assess risk factors for such infections. As a secondary objective, the clinical, laboratory and prognostic features of community-acquired endocarditis were compared. Factors related to the in-hospital lethality of culture-negative endocarditis were also assessed. Between January 2004 and January 2009, 369 consecutive cases of endocarditis were investigated in patients attending the no Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo - InCor HC-FMUSP. Cases occurring in adults, those classified by the modified Duke criteria as \"defined endocarditis\" and community-acquired cases were studied. In total, 221 cases of endocarditis comprising 170 culture-positive and 51 culturenegative cases were included. For the culture-negative cases, serology (indirect immunofluorescence reaction) and histopathological analyses for Bartonella spp. and Coxiella burnetii were performed. Cases were considered positive for Bartonella henselae or Bartonella quintana with IgG titers >= 800 and for Coxiella burnetii with antiphase I IgG titers > 800. Histopathological studies of the cardiac valves were capable of morphologically identifying the etiology in 87% of the culture-negative endocarditis cases, whereas the Gram stain was only positive in 10% of cases using fresh tissue. Culture-negative endocarditis patients presented a greater frequency of dyspnea on admission (p=0.001), lower C-reactive protein levels (p=0.009), and a lower left ventricular ejection fraction (LVEF) (p=0.022), and they required more time to start antibiotic therapy (p < 0.001) when compared with culture-positive patients. There was no statistically significant difference between the two groups regarding in-hospital lethality or survival after hospital discharge. Diabetes mellitus (p=0.01) or severe sepsis on admission (p=0.01) were independently associated with in-hospital death for culture-negative endocarditis. Ten cases of endocarditis caused by Bartonella spp. (frequency 19.6% [IC95%: 9.8 - 33.1]) and 4 caused by Coxiella burnetii (frequency 7.8% [IC95%: 2.2 - 18.9]) were diagnosed among the 51 cases of culture-negative endocarditis. Endocarditis caused by Bartonella spp. was associated with lower LVEF values (p=0.025), the identification of Gram-negative coccobacilli in cardiac valve histology (p=0.001) and the presence of a cat in the patient\'s residence (p=0.001). Conclusions: Bartonella spp. and Coxiella burnetii were the causative etiology of almost one-third (27.5%) of the community-acquired cases of culture-negative endocarditis. The presence of a cat in the patient\'s residence, a LVEF <= 45% and the identification of Gram-negative coccobacilli in the histological examination of the cardiac valve in patients with culturenegative endocarditis appear to be associated with Bartonella spp. as the causative etiology. Histological examination of the cardiac valves allowed for morphological identification of the causative microorganism in the majority of cases, even when blood cultures were negative. There was no difference in in-hospital lethality or long-term survival between the two groups. The presence of diabetes mellitus or severe sepsis at admission was associated with in-hospital death in cases of culture-negative endocarditis

Parmi les nombreuses pathologies infectieuses dignes d’intérêt en Guyane, deux d’entre elles, deux zoonoses, ont connu récemment un regain d’intérêt conduisant en quelques années à améliorer nettement leur connaissance, mais également découvrir des particularités épidémiologiques inattendues qui nous ont amené à nous poser la question de leur caractère émergent ou réémergent. Bien que cosmopolite et à tropisme tropical, la leptospirose n’a que peu été décrite en Guyane et sur le bouclier des Guyanes. La littérature repose sur des cas cliniques ou série de cas anciens, la dernière publication remontant à 1995. Sont présentées ici plusieurs études qui ont permis d’en savoir un peu plus sur cette infection bactérienne : revue exhaustive de la littérature, étude rétrospective des rapports du CNR, étude rétrospective multicentrique sur les leptospiroses prises en charge en Guyane entre 2007 et 2014, avec analyse de ses déterminants, démographiques, écologiques, cliniques, séro-épidémiologique, comparaison de ses formes graves à celles d’Afrique du Nord. Bien que sa présence ait été identifiée dès les années 50 en Guyane, la fièvre Q ou infection à Coxiella burnetii, n’avait suscité localement aucun intérêt jusqu’à la fin des années 1990. Le travail ici présente la progression des connaissances sur cette infection bactérienne, également cosmopolite, mais avec des spécificités locales tout à fait inédites. Au fil des découvertes sur cette bactérie à la sauce guyanaise, nous présenterons la contribution de notre équipe à la progression du savoir sur cette pathologie et l’apport de réponses amenant tout autant de nouvelles. Ainsi les réflexions portent autour de ce génotype si particulier, le MST17, trouvé exclusivement en Guyane, qui entraine l’incidence la plus élevée au monde de la fièvre Q, une prévalence élevée parmi les pneumopathies retrouvée nulle ailleurs. En outre, le cycle épidémiologique de la bactérie, habituellement fondé sur le bétail, semble ici suivre un tout autre chemin et trouver son réservoir dans la faune sauvage. L’on s’interroge également sur le contraste entre le problème de santé publique majeur que cette maladie représente en Guyane et le caractère tout juste anecdotique dans le reste de l’Amérique latine.Finalement, bien que ces deux zoonoses puissent être qualifiées de « maladies nouvelles » en Guyane, il s’agit probablement pour la leptospirose d’une augmentation récente du nombre de cas lié à l’amélioration des techniques diagnostiques et à la sensibilisation des médecins à cette maladie, tandis que la fièvre Q semble présenter un véritable profil émergent, avec augmentation récente de son incidence, et de nombreuses inconnues lié à un génotype très particulier.Plusieurs questions concernant ces deux infections restent encore sans réponse, et le travail est immense pour mieux comprendre les enjeux de ces deux maladies, tant à l’échelle de la Guyane qu’à celle du continent latino-américain. / Among the numerous infectious diseases of interest in French Guiana (FG), two of them, two zoonoses, have recently experienced a revival of interest leading in a few years to a marked improvement in their knowledge. Several studies allowed as well discovering unexpected epidemiological features that have led us to question their emerging or reemerging character.Although cosmopolitan and with tropical a tropism, leptospirosis has been barely described in FG and on the Guiana Shield. The literature is old and reports only clinical cases or series, the most recent publication dating back to 1995. Several studies are presented in this work which have allowed to know a little more about this bacterial infection: exhaustive review of the literature, retrospective study of the reference national center reports, a retrospective multicenter study on leptos-piroses managed in FG between 2007 and 2014, with analysis of its determinants, demographic, ecological, clinical, sero-epidemiological, and a study comparing Guianese severe forms to those of North Africa.Although its presence had been suspected as early as the 1950s in FG, Q fever or Coxiella burnetii infection had not aroused interest locally until the late 1990s. The work here presents the progression of the knowledge of this bacterial infection, also cosmopolitan, but with unusual local specificities. In the course of the discoveries around this Guianese outbreak, we will present the contribution of our team to the progression of knowledge on this pathology and the contribution of answers bringing as much new questions. Thus the discussion will focus on this particular genotype, MST17, found exclusively in FG, which results in the highest incidence of Q fever in the world, a prevalence among pneumonias never found elsewhere. Moreover, the epidemiological cycle of the bacterium, usually based on livestock, seems to follow a completely different path and find its reservoir in wildlife. We also wonder about the contrast between the major public health problems that this disease represents in FG and the anecdotal character in the rest of Latin America.Finally, although these two zoonotic diseases may be described as "new diseases" in FG, it is likely that leptospirosis presents a recent increase in the number of cases related to the improvement of diagnostic techniques and the sensitization of physicians to this disease, but without real emergence, while Q fever seems to present a true emergent profile, with a recent increase in its incidence, and many unknowns linked to a very particular genotype.Many questions concerning these two infections remain unanswered, and the work is immense to better understand the stakes of these two diseases, both on the scale of FG and that of the Amazonian region and the Latin American continent.