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.

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.

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.

- 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.