Afin de déjouer les défenses immunitaires de l’hôte et créer les niches nécessaires à leur survie, les bactéries pathogènes mettent on œuvre de nombreux mécanismes ciblant les voies de signalisation de la cellule hôte. L’un de ces mécanismes repose sur la sécrétion de protéines bactériennes dans les cellules cibles afin de moduler directement leurs réseaux de signalisation. Cependant, les signaux, les senseurs et les effecteurs impliqués dans ces régulations sont encore peu ou mal connus. La détection de l’environnement dans la cellule hôte lors de l'infection est l’élément clé d’une réponse adaptée, et les systèmes de signalisation basés sur les mécanismes de phosphorylation sont indispensables à l'adaptation hôte-pathogène. L’aspect innovant de ce projet repose sur l’étude du rôle des Ser/Thr kinases et phosphatases sécrétées lors des interactions hôte-pathogène, modifiant ainsi la réponse globale de l’hôte durant l’infection. Pendant ma thèse, j’ai tout d’abord étudié le rôle d’une nouvelle protéine kinase bactérienne identifiée chez Coxiella burnetii, nommée CstK (Coxiella serine threonine Kinase). C. burnetii, l’agent étiologique de la zoonose appelée fièvre Q, modifie les défenses de la cellule hôte, permettant sa réplication dans des vacuoles spécifiques à l’intérieur de la cellule hôte. Par ailleurs, la sécrétion d’un grand nombre d’effecteurs bactériens est indispensable au détournement du phagosome par Coxiella. Nous ainsi avons démontré que cette potentielle protéine kinase, identifiée in silico dans le génome de C. burnetii, est capable de s’autophosphoryler et par conséquent possède une activité kinase. De plus, nous avons identifié différentes protéines spécifiques de la cellule hôte interagissant avec CstK à l'aide du modèle amibe Dictyostelium discoideum, un phagocyte professionnel eucaryote, permettant des études génétiques et biochimiques. Dans la deuxième partie de mon projet, je me suis intéressée au rôle d’une probable protéine sécrétée, la tyrosine phosphatase PtpA, durant l’infection par Staphylococcus aureus. Bien connue dans les hôpitaux, où elle est responsable de nombreuses maladies nosocomiales, cette bactérie possède un grand nombre de facteurs de virulence, responsables d’infections variées, et l’apparition exponentielle de souches multi-résistantes en font un problème majeur. Ce pathogène est capable d’envahir et de persister dans un grand nombre de types cellulaires différents chez l’Homme, en sécrétant des protéines effectrices qui vont moduler les réponses cellulaires. Nous avons démontré que PtpA était sécrétée durant la phase de croissance bactérienne, et pu déterminer que PtpA possédait une activité tyrosine phosphatase, régulée par la tyrosine kinase CapA1B2 de S. aureus. Enfin, en utilisant le modèle D. discoideum, nous avons pu identifier des protéines de l’hôte qui interagissent avec PtpA, mais leur rôle dans l’infection n’est pas encore connu. / Bacterial pathogens have developed diverse strategies towards host signalling pathways, in order to subvert the immune response and/or create permissive niches for their survival. One such strategy is based on the secretion of bacterial signalling proteins into the target host cells, thereby directly modulating the status of host signalling networks. Because the mechanisms involved are largely intractable to most in vivo analyses, very little is known about the signals, sensors, and effectors mediating these adaptations. Sensing the host environment is a key component to execute appropriate developmental programs, and the eukaryotic-like phosphosignaling systems in prokaryotes are emerging as equally important regulatory systems as the well-known eukaryotic systems, but the study of their functions is still in its infancy. The innovative aspect of this project resides in the study of the emerging role of secreted Ser/Thr kinases and phosphatases in the control of host-pathogen interactions thus modifying the global host response during infection. During my thesis, I first investigated the role of a novel bacterial protein kinase identified in Coxiella burnetii that we named CstK (Coxiella serine threonine Kinase). C. burnetii, the etiological agent of the emerging zoonosis Q fever, subverts host cell defenses, permitting its intracellular replication in specialized vacuoles within host cells. Secretion of a large number of bacterial effectors into host cell is absolutely required for rerouting the Coxiella phagosome. We demonstrated that this putative protein kinase identified by in silico analysis of the C. burnetii genome is able to autophosphorylate and undergoes in vitro phosphorylation. Moreover, we identified specific host cell proteins interacting with CstK, by the use of the model amoeba Dictyostelium discoideum, an eukaryotic professional phagocyte amenable to genetic and biochemical studies. In the second part of my project, I was interested in the role of a putative secreted protein tyrosine phosphatase (PtpA) during Staphylococcus aureus infection. Well-known in hospital-acquired diseases, this bacteria produces multiple virulence factors that lead to various severe diseases, and the increase of multi-resistant strains is a major concern. This pathogen has the ability to invade and persist in a number of different human host cell types, secreting effector proteins to modulate cellular responses. Here we demonstrated that PtpA is secreted during the bacterial growth. We also determined that PtpA presents a tyrosine phosphatase activity that is regulated by the tyrosine protein kinase CapA1B2 of S. aureus. At last, using the D. discoideum model, we identified some host proteins that interact with PtpA, but their link with infection still remain to be studied.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-01-08T17:55:42Z No. of bitstreams: 1 letíciascafuttodefaria.pdf: 1095599 bytes, checksum: 1c63329ef1875b3086d3531350f36ccc (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-01-23T11:12:07Z (GMT) No. of bitstreams: 1 letíciascafuttodefaria.pdf: 1095599 bytes, checksum: 1c63329ef1875b3086d3531350f36ccc (MD5) / Made available in DSpace on 2018-01-23T11:12:07Z (GMT). No. of bitstreams: 1 letíciascafuttodefaria.pdf: 1095599 bytes, checksum: 1c63329ef1875b3086d3531350f36ccc (MD5) Previous issue date: 2017-08-22 / O objetivo desse trabalho foi estimar a prevalência de Coxiella burnetii em Queijo Minas Artesanal da microrregião do Serro e identificar os fatores associados à presença desse microrganismo nas propriedades estudadas. Foram sorteados aleatoriamente 55 produtores cadastrados no Instituto Mineiro de Agropecuária (IMA) para participar da pesquisa. Os produtores responderam a um questionário e foi coletada uma amostra de queijo de cada propriedade para representá-la. Nas amostras de queijo coletadas, foram realizadas as análises de Reação de Cadeia da Polimerase (PCR) para C. burnetii e sequenciamento dos produtos de DNA amplificados. Os dados provenientes das entrevistas e resultados das análises laboratoriais, uma vez sistematizados, formaram a base de dados do trabalho. A presença de DNA de C. burnetii foi considerada a variável dependente e as variáveis explicativas foram divididas em três grupos hierárquicos: distal (socioeconômicas e demográficas), intermediário (produção de queijo) e proximal (características do rebanho, produção de leite e ordenha). Os fatores associados à presença de C. burnetii nos queijos foram identificados por análises de regressão logística univariada e multivariada. Do total de amostras analisadas (n = 53), cinco (9,43%) apresentaram DNA de C. burnetii confirmado por sequenciamento. Os fatores associados com a presença de DNA de C. burnetii nos queijos foram: uso do pingo (soro- fermento) para a fabricação dos queijos (OR= 12,09), tipo de ordenha (OR= (16,45) e número de vacas em lactação (OR= 1,05). As variáveis que permaneceram no modelo final como explicativas para presença de C. burnetii nos queijos foram: número de vacas em lactação, tipo de ordenha e uso do pingo. A presença de DNA de C. burnetii nos queijos artesanais enfatiza, por um lado, a necessidade de medidas de controle mais rigorosas para que essa bactéria não esteja presente nos rebanhos de propriedades produtoras de queijos artesanais. Por outro lado, o conhecimento dos fatores associados à presença de C. burnetii nos queijos artesanais poderá auxiliar no estabelecimento de medidas preventivas e na criação de novas normas e legislação para a produção de queijos artesanais, já que essa lacuna na legislação seria algo para reflexão, dada a importância da febre Q para a saúde pública. / The objective of this study was to estimate the prevalence of Coxiella burnetii in Artisan Minas cheese of region of Serro and identify the factors associated with the presence of this organism in the properties studied. Were drawn at random 55 registered producers in the Instituto Mineiro de Agropecuária (IMA) to participate in the research. The producers responded to a questionnaire and was collected a sample of cheese at a property to represent her. The cheese samples collected, the analysis of reaction of polymerase chain reaction (PCR) to C. burnetii and sequencing of amplified DNA products. Data from the interviews and results of laboratory tests, once organized, form the basis of work data. The presence of DNA of C. burnetii was considered as the dependent variable and the explanatory variables were divided into three groups: hierarchical distal (socioeconomic and demographic), intermediate (cheese production) and proximally (characteristics of the herd, milk production and milk). The factors associated with the presence of C. burnetii in cheeses were identified by analysis of univariate and multivariate logistic regression. Of the total samples analyzed (n = 53), five (9.43%) presented DNA of C. burnetii confirmed by sequencing. The factors associated with the presence of DNA of C. burnetii in cheeses were: use of the pingo (serum- baking) for the manufacture of the cheeses (OR = 12.09), type of milking (OR = (16.45) and number of lactating cows (OR = 1.05). The variables that remained in the final model as explanatory for the presence of C. burnetii in cheeses were: number of lactating cows, milking and type use the pingo. The presence of DNA of C. burnetii in artisanal cheeses emphasizes, on the one hand, the need for more stringent control measures so that this bacteria is not present in flocks of artisanal cheese-producing properties. On the other hand, the knowledge of the factors associated with the presence of C. burnetii in artisanal cheeses may assist in establishing preventive measures and the creation of new standards and legislation for the production of artisanal cheeses, since this gap in the legislation would be something for reflection, given the importance of Q fever to public health.

C. burnetii est une bactérie intracellulaire strcite. Récemment, un milieu axénique, nommé ACCM 2 a été développé, et permet la culture de cette bactérie en atmosphère microaérophile. Nous avons testé si l’ajout d’acide urique dans le milieu de culture pouvait permettre une culture en milieu aérobie. Nous avons observé une croissance de C. burnetii incubée en conditions aérobies dans le milieu ACCM2 enrichi en acide urique. A Cayenne, en Guyane Française, les pneumopathies causées par C. burnetii sont fréquentes et sévères. Nous avons analysé le génome d’une souche isolée à Cayenne. Ce travail a mis en évidence une délétion de 6105 pb intéressant le gène du système de sécrétion de type 1 (T1SS). Cette réduction de génome est probablement impliquée dans l’hypervirulence des souches de Cayenne. Enfin, nous avons testé la sensibilité aux antibiotiques de 6 souches isolées à partir de patients vivant à Cayenne. Ces souches étaient toutes sensibles à la doxycycline et résistantes aux macrolides. Dans une troisième partie nous avons analysé l’apport du TEP scanner dans le diagnostic des infections à C. burnetii. 167 patients atteints d’infections à C. burnetii ont bénéficié d’un TEP scanner. Nous avons retrouvé une proportion élevée de fixations ostéo-articulaires (21) et ganglionnaires (27), et nous avons proposé de nouvelles définitions pour ces localisations. Nous avons ensuite étudié l’impact du traitement chirurgical chez les patients atteints d’infections vasculaires. Une analyse rétrospective de 86 patients atteints d’infections vasculaires a montré que la chirurgie était associée à une diminution de la mortalité à 2,5 ans et à une meilleure évolution sérologique / C. burnetii is an intracellular bacterium. Recently, an axenic medium, named ACCM 2, has been developed and allows the culture of this bacterium in a microaerophilic atmosphere. We tested if the addition of uric acid in the medium could allow an aerobic culture. We observed growth of C. burnetii incubated under aerobic conditions in the ACCM2 medium enriched with uric acid. In Cayenne, French Guiana, pneumonia caused by C. burnetii are frequent and severe. We analyzed the genome of a strain from Cayenne. This work revealed a 6105 bp deletion in the gene of the type 1 secretion system (T1SS). This genome reduction is probably involved in the hypervirulence of Cayenne strains. Finally, we tested the antibiotic suceptibility of 6 strains isolated from patients living in Cayenne. These strains were all susceptible to doxycycline and resistant to macrolides. In a third part we analyzed the contribution of PET scanner in the diagnosis of C. burnetii infections. 167 patients with C. burnetii infections benefited from a PET scan. We found a high proportion of osteo-articular (21) and lymphadenitis (27) fixations, and we proposed new definitions for these locations. We then investigated the impact of surgical treatment in patients with vascular infections. A retrospective analysis of 86 patients with vascular infections showed that surgery was associated with a lower mortality at 2.5 years and a better serological outcome

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

Orientador: Marcos Rogério André / Coorientador: José Maurício Barbanti Duarte / Banca: Karina Paes Bürger / Banca: Ana Patrícia Yatsuda Natsui / Resumo: Coxiella burnetii é uma bactéria Gram-negativa intracelular obrigatória, que além de considerado agente zoonótico causador da Febre Q em vários países do mundo, foi classificado como um potencial agente de bioterrorismo. Bovinos, ovinos e caprinos representam as fontes de infecção mais frequentemente associadas à ocorrência da enfermidade em humanos, entretanto animais selvagens também podem atuar como importantes fontes de infecção. Desta forma, o presente estudo tem como objetivo investigar a ocorrência de Coxiella burnetii em ruminantes domésticos e selvagens no Brasil. Para tal, 188 amostras de sangue de cervídeos (143 Blastocerus dichotomus, 11 Ozotocerus bezoarticus, 27 Mazama gouazobira, 4 M. bororo e 3 M. americanum), capturados nos estados de MS, SP e MG, foram submetidas à extração de DNA e, subsequentemente, à nested (n)PCR para C. burnetii baseada no elemento de inserção repetitivo IS1111 do gene heat shock protein (htpAB). Além disso, 169 amostras de soro de cervídeos foram submetidas à Reação de Imunofluorescência para detecção de anticorpos IgG anti-C. burnetii. Amostras de soros de bovinos apresentando desordens reprodutivas foram submetidas às Reações de Vírus Neutralização para BoHV-1 e BVD, Soroaglutinação Microscópica para Leptospira spp., Reação de Imunofluorescência Indireta for C. burnetii e Toxoplasma gondii, e Ensaio de Imunoabsorção Enzimática Indireto para Neospora caninum e Trypanosoma vivax. Todas as amostras de sangue mostraram-se negativas na nP... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Coxiella burnetii is an obligate intracellular Gram-negative bacterium, which, in addition to being considered a zoonotic agent that causes Q fever in several countries of the world, has been classified as a potential bioterrorism agent. Cattle, sheep and goats represent the most frequent sources of infection associated with the occurrence of the disease in humans, however, wild animals can also act as important sources of infection. In this way, the present study aims to investigate the occurrence of Coxiella burnetii in domestic and wild ruminants in Brazil. To this end, 188 cervus blood samples (143 Blastocerus dichotomus, 11 Ozotocerus bezoarticus, 27 Mazama gouazobira, 4 M. bororo and 3 M. americanum), captured in the states of MS, SP and MG, were subjected to DNA extraction and, subsequently to the nested (n) PCR for C. burnetii based on the heat shock protein (htpAB) gene IS1111 insertion element. In addition, 169 cervical serum samples were submitted to Immunofluorescence Reaction for the detection of anti-C. burnetii IgG antibodies. Adicionaly, samples of bovine sera presenting reproductive disorders were submitted to the Virus Reaction Neutralization for BoHV-1 and BVD, Microscopic Soroagglutination for Leptospira spp., Indirect Immunofluorescence Reaction for C. burnetii and T. gondii, and Indirect Enzyme Immunoabsorption Assay for N caninum and T. vivax. All blood samples were negative in nPCR, evidencing absence of circulating DNA of C. burnetii in the sampled c... (Complete abstract click electronic access below) / Mestre

Submitted by Michele Mologni (mologni@unoeste.br) on 2019-02-06T17:06:34Z No. of bitstreams: 1 Maykon Ramos Brasileiro.pdf: 195879 bytes, checksum: 8506daf0073cf47f0b9ce3020a93c843 (MD5) / Made available in DSpace on 2019-02-06T17:06:34Z (GMT). No. of bitstreams: 1 Maykon Ramos Brasileiro.pdf: 195879 bytes, checksum: 8506daf0073cf47f0b9ce3020a93c843 (MD5) Previous issue date: 2018-10-31 / . / A coxielose é uma zoonose associada à bactéria intracelular Coxiella burnetii sendo uma infecção comum em ruminantes e humanos de países asiáticos e europeus. No Brasil estudos epidemiológicos sobre a enfermidade são escassos. O presente estudo avaliou a presença de anticorpos anti-Coxiella (fase II) em 231 amostras de soro bovino provenientes de doze fazendas de leite e corte da região Oeste do estado de São Paulo, colhidas entre os anos de 2012-2018, que estavam mantidas em estoque sob congelamento a -25oC, utilizadas em outras rotinas diagnósticas. As amostras foram testadas pelo método ELISA, utilizando kit comercial importado (Bio-X - Diagnostics®). Do total avaliado, 176 (76,2%) animais foram sorologicamente positivos. Onze (91,6%) das 12 propriedades apresentaram ao menos um animal positivo, com prevalências variando entre 20 e 100%. Os resultados denotam alta prevalência da coxielose em bovinos de leite e corte na região Oeste Paulista, sugerindo que a enfermidade deve ser avaliada com relação aos potencias impactos na reprodução dos animais, incluindo diagnóstico diferencial com patógenos abortivos. Considerando-se o risco de transmissão zoonótica e o desconhecimento acerca da doença, as autoridades de saúde locais devem estar atentas a possíveis casos humanos subdiagnosticados ou confundidos com outras enfermidades similares.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Ruminants are considered the commonest animal reservoir for human infection of Coxiella burnetii, the Q fever causative agent. Considering the recently described importance of human Q fever in Greece, we aimed at providing the first comprehensive direct evidence of C. burnetii in dairy cows in Greece, including the genetic characterization of strains. The 462 examined dairy farms represented all geographical areas of Greece. One bulk tank milk sample was collected from every farm and tested for the presence of C. burnetii. Molecular genotyping of strains, performed directly on samples, revealed the existence of two separate clades characterized by single nucleotide polymorphism (SNP) genotypes of type 1 and type 2. The two clades were clearly distinguished in multiple locus variable-number tandem repeat analysis (MLVA) by two discriminative loci: MS30 and MS28. Whereas MLVA profiles of SNP-type 2 clade were closely related to strains described in other European cattle populations, the MLVA profile observed within the SNP type 1 clade highlighted a peculiar genetic signature for Greece, related to genotypes found in sheep and goats in Europe. The shedding of C. burnetii bearing this genotype might have yet undefined human epidemiological consequences. Surveillance of the genetic distribution of C. burnetii from different sources is needed to fully understand the epidemiology of Q fever in Greece.

- Einleitung - Coxiella burnetii (C. burnetii) ist der Erreger des Q-Fiebers, einer zoonotischen Erkrankung, welche insbesondere kleine und große Wiederkäuer betrifft. Während Tiere meist asymptomatisch erkranken, kann die Erkrankung beim Menschen von akuten grippeähnlichen Symptomen bis hin zur chronischen Organschädigung führen. Die Diagnostik von Q-Fieber im veterinärmedizinischen Bereich wird durch unspezifische Symptome sowie diagnostische Lücken erschwert: Einerseits ist die direkte Diagnostik aufgrund langer Anzuchtzeiten der Bakterien häufig unpraktikabel. Andererseits weisen die Leistungen der Serodiagnostika in Untersuchungen teilweise inakzeptable Spezifitäten und Sensitivitäten auf. - Ziel der Untersuchungen - Ziel war es, das Potential des rekombinant hergestellten C. burnetii Außenmembran-proteins Com1 als diagnostischen Marker zu bestimmen und darauf aufbauend einen indirekten Enzyme-linked Immunosorbent Assay (ELISA) zu entwickeln, welcher die Grundlage für neue, praxistaugliche und verbesserte Serodiagnostika bieten kann. - Material und Methoden - Hierfür wurde die kodierende Sequenz für Com1 von C. burnetii Nine Mile Phase II RSA 439 unter Ausschluss der Signalsequenz mittels Polymerase-Kettenreaktion amplifiziert. Das Amplifikat com1 wurde in ein Expressionsplasmid kloniert und anschließend mittels Transformation in Escherichia coli (E. coli) als Com1 exprimiert. Das Expressionsprodukt wurde nach der Aufreinigung über Nickel-NTA (Nitrilotriessigsäure) mittels SDS-PAGE (Natriumdodecylsulfat-Polyacrylamid-Gelelektrophorese), Coomassie-Blau-Färbung und Western Blot überprüft. Die 96-Well-Mikrotiterplatten wurden mit 1 µg/Well des rekombinanten Proteins beschichtet und 404 Seren von Schafen (111), Ziegen (100) und Rindern (193) auf ihre Reaktion im rekombinanten Com1-ELISA analysiert. Die Seren stammten sowohl aus definierten Ausbrüchen als auch aus Q-Fieber-freien Beständen, geimpften Herden und von Tieren mit fraglichem Infektionshintergrund. Daneben wurden die Seren mit kommerziellen ELISAs getestet und daraufhin in positive und negative Seren eingeteilt. Die OD450-Werte (optische Dichte bei 450 nm) der Seren aus dem rekombinanten Com1-ELISAs wurden in Pivot-Tabellen angeordnet und anschließend in einer ROC-Kurve (receiver operating characteristic) dargestellt, wodurch das Integral als Area under the curve (AUC) berechnet und hinsichtlich der Diskrimination zwischen positiven und negativen Ergebnissen ausgewertet wurde. Nach Festlegung von tierart-spezifischen Cut-off-Werten wurden Sensitivität und Falsch-Positiv-Rate bestimmt. - Ergebnisse - Die ermittelten tierart-spezifischen OD450-Cut-off-Werte betrugen für Schafe 0,32, für Ziegen 0,23 und für Rinder 0,18. Als Spezifität bzw. Sensitivität ergaben sich für Schafe 85 % bzw. 68 %, für Ziegen 94 % bzw. 77 % und für Rinder 71 % bzw. 70 %. Die Diskrimination wurde bei Schafen als „exzellent“, bei Ziegen als „herausragend“ und bei Rindern als „akzeptabel“ eingeschätzt. - Schlussfolgerungen - Die Ergebnisse dieser Studie zeigen, dass das Coxiella-Außenmembranprotein Com1 als Basis für die Entwicklung neuer, sensitiverer und spezifischerer (Schnell)-Diagnostika dienen kann, um weitere Q-Fieber-Epidemien einzudämmen und damit das Risiko einer C. burnetii-Infektion für Mensch und Tier zu minimieren. / - Introduction - Coxiella burnetii (C. burnetii) is the causative agent of Q fever, a zoonotic disease with small and large ruminants as the target hosts. While an infection in ruminants is often symptomless, the disease in humans can lead from acute flu-like symptoms to chronic organ damage. In addition to the non-specific symptoms in the veterinary field, for which reason it is difficult to directly conclude on a Q fever outbreak, the detection of infections with C. burnetii is in need of improvement. On the one hand, direct diagnosis is often impracticable due to long cultivation periods of the bacteria. On the other hand, the performance of serodiagnostics shows partially unacceptable specificities and sensitivities. - Objectives - The aim of the study was to determine the potential of the recombinantly produced C. burnetii outer membrane protein Com1 as a diagnostic marker and, subsequently, to develop an indirect enzyme-linked immunosorbent assay (ELISA), which can provide the basis for the development of new and improved serodiagnostics. - Material and methods - For this purpose, primers for com1 were designed in silico and the open reading frame (ORF) was amplified by polymerase chain reaction (PCR) using phenol-chloroform-isolated DNA (deoxyribonucleic acid) from C. burnetii Nine Mile Phase II RSA 439 without signal sequence. After control by gel electrophoresis, the amplified product was cloned into the two-stage gateway system (Invitrogen) and transformed into Escherichia coli (E. coli) TOP10. The expression vector was cloned into E. coli BL21(DE3) and Com1 was expressed. The protein was analyzed after native purification via Nickel-NTA (nitrilotriacetic acid) by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), Coomassie blue staining and Western blot and subsequently concentrated. Microtiter plates were coated with the recombinant protein and 404 sera from sheep (111), goats (100) and cattle (193) were tested for their reaction in the recombinant Com1-ELISA. The sera were derived from 36 different flocks, both from defined outbreaks and from Q fever-free flocks, vaccinated herds and from animals with questionable infection background. In addition, the sera were tested with commercial ELISAs and could thus be divided into positive and negative sera. The OD450 values (optical density at 450 nm) of the sera from the recombinant Com1 ELISAs were arranged in pivot tables and then plotted on a ROC curve (receiver operating characteristic), allowing the integral to be calculated as the area under the curve (AUC) and evaluated regarding the discrimination between positive and negative results. After establishing animal species-specific cut-off values, sensitivity and false positive rate were determined. - Results - The animal species-specific OD450 cut-off values determined were 0.32 for sheep, 0.23 for goats and 0.181 for cattle. Specificity and sensitivity were 85 % and 68 % for sheep, 94 % and 77 % for goats and 71 % and 70 % for cattle. Discrimination was assessed as 'excellent' in sheep, 'outstanding' in goats and 'acceptable' in cattle. - Conclusions - The results of this study suggest that the Coxiella outer membrane protein Com1, together with other immunogenic marker proteins, may serve as a basis for the development of new more sensitive and specific diagnostic tools to contain further Q fever epidemics and thus minimize the risk of C. burnetii infection for humans and animals. Com1 could also lay the foundation for direct rapid diagnostics.

Coxiella burnetii est une bactérie zoonotique affectant un grand nombre d’espèces animales. Chez les ruminants domestiques, l’infection est généralement asymptomatique, mais parfois associée à des problèmes reproducteurs. Néanmoins, le cycle de transmission de l’infection chez ceux-ci demeure peu connu. Dans ce contexte, nous avons réalisé une étude auprès de fermes bovines, ovines et caprines dans deux régions administratives du Québec afin d’estimer les prévalences de cette infection et d’identifier les facteurs de risque, aux niveaux individuel et troupeau, associés à la positivité. Nous avons estimé une prévalence de positivité au niveau troupeau de 44.6 % (IC95%=33.0-56.6) chez les bovins, de 70.8 % (IC95% =48.9-87.4) chez les ovins et de 66.7 % (IC95% =22.3-95.7) chez les caprins. Une association a été observée chez les troupeaux bovins entre leur positivité et la densité de petits ruminants par kilomètre carré dans un rayon de cinq kilomètres entourant la ferme. Chez les petits ruminants, une association avec la positivité des troupeaux a été observée avec la taille des troupeaux et la présence d’un chien sur la ferme. Au niveau individuel, le nombre de jours en lait ainsi que l’âge des petits ruminants étaient associés à la positivité, et ce dernier facteur était modulé par l’accès des animaux au pâturage. Aucun agrégat spatial de fermes positives n’a été détecté chez aucune des trois espèces. L’infection par Coxiella burnetii est donc fréquente dans les troupeaux de ruminants domestiques québécois et semble associée à certaines pratiques de régie et à la présence, ou proximité, d’autres animaux domestiques. / Coxiella burnetii is a zoonotic bacteria affecting a vast range of animal species. In domestic ruminants, the infection is usually asymptomatic, but sometimes linked with reproductive disorders. However, the transmission cycle of infection among them remains unclear. In that context, we conducted a study among dairy cattle, sheep and goats farms in two administrative regions of Québec to estimate the infection prevalence and identify the risk factors associated with farms and animals positivity. We estimated a herd prevalence of 44.6 % (95%CI=33.0 to 56.6) in dairy cattle, 70.8 % (95%CI=48.9 to 87.4) in sheep and 66.7 % (95%CI=22.3-95.7) in goats. On dairy cattle farms, we observed an association between their positivity and the density of small ruminants per square kilometer within a five kilometers radius around the farm. In small ruminants, at herd level, we observed an association with positivity and herd’s size and the presence of a dog on the farm. At the individual level, an association with positivity was found with the number of days in milk for small ruminants and their age, but the latter was also modulated by the individual’s previous access to pasture. No spatial cluster of positive farms was detected significant among dairy cattle nor small ruminants. The infection by Coxiella burnetii is therefore common on dodmestic ruminants’ farms in Québec and associated with some farm management practices and the presence, or proximity, of other domestic animals.