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The intestinal tract of virtually any metazoan, including mammals, is colonized with a complex microbial community to which we refer as intestinal or gut microbiota. One of the roles of the healthy intestinal microbiota is to protect the host against gut colonization with pathogenic bacteria through a phenomenon known as colonization resistance (CR). Dysbiosis of the intestinal microbiota, usually as a result of an antibiotic treatment, may lead to the disruption of the CR, and subsequent colonization with bacterial pathogens. However, and despite the importance, the role of the microbiota dysbiosis on the gut colonization by many bacterial pathogens, such as multidrug resistant Enterobacteriaceae, has not been elucidated: the members of the microbiota that confer CR and factors that promote colonization remain mostly unknown.The general aim of this thesis has been to improve the understanding of the role of the microbiota dysbiosis in gut colonization by bacterial pathogens. For this purpose, 3 projects have been established. In the first project we tried to elucidate the role of the microbiota dysbiosis on colonization by multidrug resistant Enterobacteriaceae (MRE) in mice. In the second project we investigated the risk factors and members of the microbiota associated with the MRE colonization in hospitalized patients. MRE infections represent a great threat for hospitalized patients. Specifically, acute leukemia patients are often colonized with MRE, probably due to the impaired CR as a result of intensive antibiotic treatments these patients receive. In the third project we studied the role of the microbiota dysbiosis on the development of Epizootic Rabbit Enteropathy (ERE). ERE is a severe gastrointestinal disease with a high percentage of mortality that occurs in young rabbits during first weeks post-weaning. ERE rabbits have been shown to suffer microbiota dysbiosis during the development of the disease. Moreover, the disease could be reproduced by contact between healthy and sick animals and by administration of cecal contents from ERE rabbits to healthy rabbits, suggesting that a pathogenic agent may be involved in the development of this intestinal pathology, although no causative agent has been identified until now., El tracto intestinal de prácticamente cualquier metazoo, incluidos los mamíferos, está colonizado por una compleja comunidad microbiana a la que nos referimos como microbiota intestinal. Uno de los papeles de la microbiota intestinal es proteger al huésped contra la colonización intestinal con bacterias patógenas a través de un fenómeno conocido como resistencia a la colonización (RC). La disbiosis de la microbiota intestinal, a menudo como resultado de un tratamiento antibiótico, puede conducir a la alteración de la RC y posterior colonización por patógenos bacterianos. Sin embargo, y pese a su importancia, el papel de la disbiosis de la microbiota en la colonización intestinal por muchos patógenos bacterianos, como son las Enterobacterias multirresistentes, no se ha esclarecido: los miembros de la microbiota que confieren RC y los factores que promueven la colonización siguen siendo desconocidos.El objetivo general de esta tesis ha sido mejorar la comprensión del papel de disbiosis de la microbiota en la colonización intestinal por patógenos bacterianos. Para ello se han establecido tres proyectos. En el primer proyecto investigamos el papel de disbiosis de la microbiota intestinal en la colonización por Enterobacterias multiresistentes (MRE) en ratones. En el segundo proyecto investigamos los factores de riesgo y los miembros de la microbiota asociados con la colonización por MRE en pacientes hospitalizados. Las infecciones por MRE representan una gran amenaza para los pacientes hospitalizados. Específicamente, MRE a menudo colonizan los pacientes con leucemia aguda, probablemente debido a que la RC está alterada como resultado de tratamientos antibióticos intensivos recibidos por estos pacientes. En el tercer proyecto investigamos el papel de la disbiosis microbiana en desarollo de Enteropatía Epizoótica de Conejo (ERE). ERE es una enfermedad gastrointestinal severa con un alto porcentaje de mortalidad que ocurre en conejos jóvenes durante las primeras semanas después del destete. Se ha demostrado que los conejos con ERE sufren disbiosis microbiana después del inicio de la enfermedad, aunque no está claro el papel de la disbiosis en el desarollo de la enfermedad. Además, la enfermedad puede ser reproducida por contacto entre animales sanos y enfermos y por la administración del contenido cecal de conejos con ERE a conejos sanos, lo que sugiere que un agente patógeno podría estar implicado en el desarrollo de esta patología intestinal, aunque hasta ahora no se ha logrado identificar ningún agente causal., El tracte intestinal de pràcticament qualsevol metazoo, inclosos els mamífers, està colonitzat per una complexa comunitat microbiana a la qual ens referim com microbiota intestinal. Un dels papers de la microbiota intestinal és protegir a l'hoste contra la colonització intestinal amb bacteris patògens a través d'un fenomen conegut com a resistència a la colonització (RC). La disbiosis de la microbiota intestinal, frecuentment com a resultat d'un tractament antibiòtic, pot conduir a l'alteració de la RC i posterior colonització per patògens bacterians. No obstant això, i malgrat la seva importància, el paper de la disbiosis de la microbiota en la colonització intestinal per molts patògens bacterians, com són les Enterobacteries multirresistentes, no s'ha esclarit: els membres de la microbiota que confereixen RC i els factors que promouen la colonització segueixen sent desconeguts.L'objectiu general d'aquesta tesi ha estat millorar la comprensió del paper de la disbiosis de la microbiota en la colonització intestinal per patògens bacterians. Per a això s'han establert tres projectes. En el primer projecte vam investigar el paper de la disbiosis de la microbiota intestinal en la colonització per Enterobacteries multiresistentes (MRE) en ratolins. En el segon projecte, investiguem els factors de risc i els membres de la microbiota associats amb la colonització per MRE en pacients hospitalitzats. Les infeccions per MRE representen una gran amenaça per als pacients hospitalitzats. Específicament, MRE sovint colonitza els pacients amb leucèmia aguda, probablement a causa de que la RC està alterada com a resultat de tractaments antibiòtics intensius rebuts per aquests pacients. En el tercer projecte, vam investigar el paper de la disbiosis microbiana en desenvolupament de l'Enteropatía Epizoótica de Conill (ERE). ERE és una malaltia gastrointestinal severa amb un alt percentatge de mortalitat que ocorre en conills joves durant les primeres setmanes després del deslleti. S'ha demostrat que els conills amb ERE sofreixen disbiosis microbiana després de l'inici de la malaltia, encara que no és clar el paper de la disbiosis en el desenvolupament de la malaltia. A més, la malaltia pot ser reproduïda per contacte entre animals sans i malalts i per l'administració del contingut cecal de conills amb ERE a conills sans, la qual cosa suggereix que un agent patogen podria estar implicat en el desenvolupament d'aquesta patologia intestinal, encara que fins ara no s'ha aconseguit identificar cap agent causal., Djukovic, A. (2017). Role of intestinal dysbiosis on gut colonization by bacterial pathogens [Tesis doctoral no publicada]. Universitat Politècnica de València. doi:10.4995/Thesis/10251/90415, TESIS

International audience; Epizootic rabbit enteropathy (ERE), a highly lethal (30-80% mortality) disease of broiler rabbits aged 6-14 weeks, first appeared in 1997 in French intensive enclosed rabbitries and is of unknown aetiology. Bacteriological, virological and parasitical examination of the intestinal contents of rabbits that had died either in spontaneous field cases or after experimental reproduction of ERE, were undertaken in an attempt to identify infectious agents that may play a role in the disease. Two bacterial strains, Clostridium perfringens and non-enteropathogenic Escherichia coli, were repeatedly isolated at high faecal counts from naturally infected animals. In field cases, a correlation between typical gross lesions of epizootic enteropathy and the presence of the alpha toxin of Cl. perfringens was observed (P < 0.0001; Chi-squared test). Although attempts to reproduce the disease by inoculation with different pools of cultivable bacterial strains failed, the disease was successfully reproduced by inoculation with one French and two Belgian samples of caecal contents. (c) 2005 Elsevier Ltd. All rights reserved.

International audience; In late 1996 in France, a severe digestive disease appeared in fattening domestic rabbits. Named the Epizootic Rabbit Enteropathy (ERE), this digestive syndrome has become the main cause of mortality in rabbit farming. The diagnosis in field conditions is difficult because co-infection with other common rabbit pathogens is frequent. By using specific pathogenic free (SPF) rabbits and starting from a field sample of intestinal contents of diseased animals, a virulent material (inoculum) was obtained free of almost all known pathogens but reproduced the symptoms and lesions of ERE. Four hundred and seven SPF rabbits were used in five trials to describe the disease. ERE is characterized by a high contagiousness, 30 to 40% mortality in a few days and about 100% morbidity whatever the dose of the inoculum used. Clinical signs and lesions evolved acutely with the first sign (rambling noise) appearing one day after inoculation and the disease peaking 4 to 6 days later. Growth was strongly lowered from the second day to the end of the second week. Rambling noise and distended abdomen were frequent, mucus excretion and cecal impaction were frequent but not constant. ERE at necropsy was characterized by the absence of any inflammatory or congestive lesions on the gut or on other organs but with the typical presence of a stomach and/or duodenum dilated by liquid and gas and by the absence of specific histological lesions. The etiological agent has not been identified yet, but we demonstrate that the intestinal content was infectious as early as the second day. This work constitutes the experimental basis for studies on this emerging disease within the framework of etiological research led in different European laboratories working with the infectious material.

[EN] Digestive diseases are the main cause of morbidity and mortality in growing rabbits. Clostridium perfringens is a widely occurring pathogenic bacterium in enteric diseases of domestic animals and its pathogenicity stems from the production of potent exotoxins. This work aimed to quantify the concentration of C. perfringens [alfa]-toxin in caecal samples of rabbits with digestive diseases and relate these concentrations to C. perfringens counts. Additionally, C. perfringens strains isolated from rabbits with clinical lesions of Epizootic Rabbit Enteropathy (ERE) were toxinotyped. To conduct this work, a total of 711 rabbits weaned at 35 days were housed in pairs and fattened until the age of 63 days. No experimental infection was performed and no antibiotics were provided in the feed or drinking water. All rabbits displaying symptoms of digestive diseases were slaughtered and necropsied. At 46 days, 88 healthy rabbits were also slaughtered. Caecal contents were sampled from all slaughtered animals. Thirty-seven out of the 69 rabbits with digestive diseases (88.5% of sick animals) showed ERE-confirmed symptoms and lesions. Apart from diarrhoea, the most constant ERE signs were abdominal bloating and borborygmi. At necropsy, the anterior digestive tract was found filled with large amounts of gas and liquid. Twenty-seven rabbits had liquid caecal contents whereas caecal impaction only appeared in 10 rabbits. Live weight was lower by 49.2% (P, The first author sincerely thanks the Universidad Politécnica de Madrid for funding his stay at Dr. Ducatelle’s laboratory in Universiteit Gent where he was trained in culture-based microbiology and toxin analyses. This work was supported by CICYT project AGL2008-00627.

International audience; Epizootic rabbit enteropathy is a gastrointestinal disorder of unknown aetiology of farmed rabbits characterised by inanition and mortality. Genomic analyses of virulent and non-irulent samples of inocula were performed using random amplification of polymorphic DNA RAPD). Differences in bacterial DNA composition were found between inocula, but specific sequences were not linked with field cases of epi-zootic rabbit enteropathy.

International audience; Epizootic rabbit enteropathy is an emerging disease that has appeared in French intensive enclosed rabbit farms since the beginning of 1997. Common clinical signs are mild watery diarrhoea with considerable distension of the abdomen. At necropsy, a significant dilation of the stomach and small intestine without gross evidence of acute or chronic enteric lesions (inflammation or congestion) was observed. The purpose of this study was to describe the anatomopathologic changes concerning the small intestine and those concerning the blood profile, in experimentally infected rabbits. In a first part of the experiment, thirty animals were inoculated with a reference inoculum and five were kept as controls for clinical signs examination and histopathological study. In a second part, 17 out of the inoculated rabbits and the 5 controls animals were randomly assigned to blood testing. Microscopic lesions were studied in sections from the different parts of the small intestine (duodenum, jejunum and ileum) by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The histological findings revealed only limited inflammation in inoculated animals. Major villous changes were atrophy, fusion, destruction and loss of epithelial cells. In inoculated rabbits, the congestion and dilation of blood vessels of jejunal lamina propria were significantly higher than in control animals (P

[EN] The present study evaluated under field conditions the efficacy of bacitracin zinc soluble powder (Bacivet S(R) ) for the treatment of Epizootic Rabbit Enteropathy (ERE). A blinded study was conducted in compliance with the "Good Clinical Practice" guidance. The trial site was known to be chronically infected with ERE. A total of 384 weaned rabbits (30 days of age) were enrolled in the study. They were randomly distributed over 6 rows of cages located in the same experimental room. Two rows of cages remained untreated (control group), another 2 rows were treated with Bacivet S(R) for 14 days (T14) and 2 rows were treated with Bacivet S(R) for 21 days (T21). The medication was administered per row via the drinking water and initiated when at least one animal, showing clinical signs of ERE, died. The dose was adjusted daily according to the weight of the rabbits to achieve the target dose of 420 IU/kg body weight in the two treatment groups. Animals were observed daily for mortality, diarrhoea, tympany, or any other abnormal sign for 42 days post enrollment. Weights of the animals and feed intake were recorded on a weekly base. On the second day after the set-up of the trial, mortality already occurred with clear signs of ERE and the medication was initiated. On the primary evaluation parameter, mortality, both treated groups showed significant (P

[EN] The effect of three levels of mannan oligosaccharides (MOS at 0.5, 1.0 and 1.5 g/kg diet) compared to dietary antibiotic supplementation (ANT, colistin sulphate 144, tylosin 100 and oxytetracycline 1000 ppm) and to a diet without MOS or medication (control group) were studied on 5 groups of rabbits (n=342 cages/group and 2 rabbits/cage) from weaning (35 d) to 60 d of age in a farm with an anamnestic history of Epizootic Rabbit Enteropathy (ERE). Mortality rate was recorded daily. Thirty two cages per group were randomly chosen to record growth performance and rectal anaerobic microbial population. Live weight and feed intake were recorded weekly. At 49 d of age rectal swabs were collected with cotton swabs from 8 rabbits per group and the anaerobic microbial population was counted. Feed digestibility was determined per cage using acid insoluble ash as an internal marker, collecting faeces from 57 to 59 d of age. An episode of ERE occurred during the second and the third week of the trial. The control group showed a high mortality rate during the fattening period (78%) and was not considered for further measurements. Total mortality rate was higher (P

[EN] The aetiology of epizootic rabbit enteropathy (ERE) is still unknown despite ten years of continuous research. A putative bacterial aetiology is the basis of current research. The fractionation of the reference inoculum (TEC4) is a major step towards finding the potential bacterial agent(s). In this study, TEC4 was fractionated by different techniques: centrifugation on discontinuous sucrose gradient, cell adherence and chloroform/ethanol treatment. The different fractions were inoculated into SPF rabbits and analyzed with classical bacteriological techniques. ERE was reproduced with two of the six fractions obtained. Four species never previously cultured from TEC were identified in the process but, to date, none of them seems to be the aetiology of ERE., This work was supported by a Grant from the «Service Publique Fédéral Santé Publique, sécurité de la chaîne alimentaire et environnement: Division Recherche contractuelle», contract RT 06/7 MINRABBIT. N. Huybens is a PhD fellow of the “Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture” (F.R.I.A.).