Morfologia dos cecos de pintos de corte submetidos ao tratamento com microbiota cecal antes e após infecção por Salmonella enteritidis

Pós-graduação em Medicina Veterinária - FCAV

Influence du microbiote intestinal sur le métabolisme et la virulence des Escherichia coli entérohémorragiques

Les Escherichia coli entérohémorragiques (EHEC) représentent un problème majeur de santé publique dans les pays développés. Les EHEC sont régulièrement responsables de toxi-infections alimentaires graves chez l’humain et causent des colites hémorragiques et le symptôme hémolytique et urémique, mortel chez les enfants en bas âge. Les EHEC les plus virulents appartiennent au sérotype O157:H7 et le bovin constitue leur réservoir naturel. À ce jour il n’existe aucun traitement pour éviter l’apparition des symptômes liés à une infection à EHEC. Par conséquent, il est important d’augmenter nos connaissances sur les mécanismes employés par le pathogène pour réguler sa virulence et coloniser efficacement la niche intestinale. Dans un premier temps, l’adaptation de la souche EHEC O157:H7 EDL933 à l’activité métabolique du microbiote intestinal a été étudiée au niveau transcriptionnel. Pour se faire, EDL933 a été cultivée dans les contenus caecaux de rats axéniques (milieu GFC) et dans ceux provenant de rats colonisés par le microbiote intestinal humain (milieu HMC). Le HMC est un milieu cécal conditionné in vivo par le microbiote. Dans le HMC par rapport au GFC, EDL933 change drastiquement de profile métabolique en réponse à l’activité du microbiote et cela se traduit par une diminution de l’expression des voies de la glycolyse et une activation des voies de l’anaplérose (voies métaboliques dont le rôle est d’approvisionner le cycle TCA en intermédiaires métaboliques). Ces résultats, couplés avec une analyse métabolomique ciblée sur plusieurs composés, ont révélé la carence en nutriments rencontrée par le pathogène dans le HMC et les stratégies métaboliques utilisées pour s’adapter au microbiote intestinal. De plus, l’expression des gènes de virulence incluant les gènes du locus d’effacement des entérocytes (LEE) codant pour le système de sécrétion de type III sont réprimés dans le HMC par rapport au GFC indiquant la capacité du microbiote intestinal à réprimer la virulence des EHEC. L’influence de plusieurs composés intestinaux présents dans les contenus caecaux de rats sur l’expression des gènes de virulence d’EDL933 a ensuite été étudiée. Ces résultats ont démontré que deux composés, l’acide N-acétylneuraminique (Neu5Ac) et le N-acétylglucosamine (GlcNAc) répriment l’expression des gènes du LEE. La répression induite par ces composés s’effectue via NagC, le senseur du GlcNAc-6-P intracellulaire et le régulateur du catabolisme du GlcNAc et du galactose chez E. coli. NagC est un régulateur transcriptionnel inactivé en présence de GlcNAc-6-P qui dérive du catabolisme du Neu5Ac et du transport GlcNAc. Ce travail nous a permis d’identifier NagC comme un activateur des gènes du LEE et de mettre à jour un nouveau mécanisme qui permet la synchronisation de la virulence avec le métabolisme chez les EHEC O157:H7. La concentration du Neu5Ac et du GlcNAc est augmentée in vivo chez le rat par le symbiote humain Bacteroides thetaiotaomicron, indiquant la capacité de certaines espèces du microbiote intestinal à relâcher les composés répresseurs de la virulence des pathogènes. Ce travail a permis l’identification des adaptations métaboliques des EHEC O157:H7 en réponse au microbiote intestinal ainsi que la découverte d’un nouveau mécanisme de régulation de la virulence en réponse au métabolisme. Ces données peuvent contribuer à l’élaboration de nouvelles approches visant à limiter les infections à EHEC.

Prostacyclin, not only nitric oxide, is a mediator of the vasorelaxation induced by acetylcholine in aortas from rats submitted to cecal ligation and perforation (CLP)

Nitric oxide has been pointed out as the main agent involved in the vasodilatation, which is the major symptom of septic shock. However, there must be another mediator contributing to the circulatory failure observed in sepsis. This study aimed to investigate the endothelium-dependent relaxation induced by acetylcholine and the factors involved in this relaxation, using aortic rings isolated from rats submitted to cecal ligation and perforation (CLP), 2 h after induction of sepsis, which characterizes the hyperdynamic phase of sepsis. Under inhibition of constitutive NO-synthases (cNOS), the relaxation induced by acetylcholine was greater in the aortic rings of rats submitted to CLP compared with sham-operated rat aortic rings. The cyclooxygenase inhibitor indomethacin normalized this response, and the concentration of the stable metabolite of prostacyclin in the aorta of CLP rats increased in basal conditions and after stimulation with acetylcholine. Acetylcholine-induced NO production was lower in the endothelial cells from the aorta of CLP rats compared with sham rat aorta, but the protein expression of the cNOS was not altered. Moreover, iNOS protein expression could not be detected. Therefore, prostacyclin, and not only nitric oxide, is a mediator of the vasorelaxation induced by acetylcholine in aortas from rats submitted to CLP. (C) 2011 Elsevier Inc. All rights reserved.

Description of a New Model of Intestinal Denervation and In Situ Ischemia-Reperfusion Injury Using the Cecal Artery for Perfusion

Background. The main purpose of the present investigation was to describe a model of intestinal denervation and in situ intestinal ischemia-reperfusion injury in adult rats, with utilization of the distal branch of the superior mesenteric artery close to the cecum for perfusion. Methods. In the root of the mesentery, the mesenteric artery and vein were completely isolated. Close to the cecal valve, a lymphatic node served as the reference point for the localization of the cecal artery, which was cannulated for perfusion with cold lactated Ringer`s solution. One hundred adult male rats were utilized in the study. Results. In a pilot study, we demonstrated that the cold ischemia time was sufficient to promote histopathologic intestinal changes characteristic of ischemia-reperfusion injury. Among 88 operated animals, 62 (70.5%) survived the procedure. Conclusion. The experimental model described herein has the advantage of preserving the entire intestine, which makes it more suitable for studies of physiological and morphological alterations after intestinal transplantation.

Treatment with cannabidiol reverses oxidative stress parameters, cognitive impairment and mortality in rats submitted to sepsis by cecal ligation and puncture

Oxidative stress plays an important role in the development of cognitive impairment in sepsis. Here we assess the effects of acute and extended administration of cannabidiol (CBD) on oxidative stress parameters in peripheral organs and in the brain, cognitive impairment, and mortality in rats submitted to sepsis by cecal ligation and perforation (CLP). To this aim, male Wistar rats underwent either sham operation or CLP. Rats subjected to CLP were treated by intraperitoneal injection with ""basic support"" and CBD (at 2.5, 5, or 10 mg/kg once or daily for 9 days after CLP) or vehicle. Six hours after CLP (early times), the rats were killed and samples from lung, liver, kidney, heart, spleen, and brain (hippocampus, striatum, and cortex) were obtained and assayed for thiobarbituric acid reactive species (TBARS) formation and protein carbonyls. On the 10th day (late times), the rats were submitted to the inhibitory avoidance task. After the test, the animals were killed and samples from lung, liver, kidney, heart, spleen, and brain (hippocampus) were obtained and assayed for TBARS formation and protein carbonyls. The acute and extended administration of CBD at different doses reduced TBARS and carbonyl levels in some organs and had no effects in others, ameliorated cognitive impairment, and significantly reduced mortality in rats submitted to CLP. Our data provide the first experimental demonstration that CBD reduces the consequences of sepsis induced by CLP in rats, by decreasing oxidative stress in peripheral organs and in the brain, improving impaired cognitive function, and decreasing mortality. (C) 2010 Elsevier B.V. All rights reserved.

Ischemic cecal perforation secondary to ESWL of junctional stone in ureterosigmoidostomy (Mainz Pouch II).

We report an unusual case of ischemic cecal perforation after extracorporeal shock wave lithotripsy for an impacted stone at the distal end of the right ureter of a ureterosigmoidostomy in a 78-year-old female patient.

Tabagisme et système digestif: une relation complexe. Partie 2: Microbiote intestinal et tabagisme [Smoking and digestive tract: a complex relationship. Part 2: Intestinal microblota and cigarette smoking].

Le système digestif est colonisé dès la naissance par une population bactérienne, le microbiote, qui influence le développement du système immunitaire. Des modifications dans sa composition sont associées à des pathologies comme l'obésité et les maladies inflammatoires chroniques de l'intestin. Outre les antibiotiques, des facteurs environnementaux comme le tabagisme semblent aussi avoir une influence sur la composition de la flore intestinale, pouvant en partie expliquer la prise de poids à l'arrêt du tabac avec une modification de la composition du microbiote proche de celle observée chez des personnes obèses (profil microbiotique montrant des capacités accrues d'extraction calorique des aliments ingérés). Ces découvertes permettent d'imaginer de nouvelles approches diagnostiques et thérapeutiques via la régulation de ce microbiome. The digestive tract is colonized from birth by a bacterial population called the microbiota which influences the development of the immune system. Modifications in its composition are associated with problems such as obesity or inflammatory bowel diseases. Antibiotics are known to influence the intestinal microbiota but other environmental factors such as cigarette smoking also seem to have an impact on its composition. This influence might partly explain weight gain which is observed after smoking cessation. Indeed there is a modification of the gut microbiota which becomes similar to that of obese people with a microbiotical profile which is more efficient to extract calories from ingested food. These new findings open new fields of diagnostic and therapeutic approaches through the regulation of the microbiota.

Behavioral deficits in sepsis-surviving rats induced by cecal ligation and perforation

Sepsis and its complications are the leading causes of mortality in intensive care units, accounting for 10-50% of deaths. Intensive care unit survivors present long-term cognitive impairment, including alterations in memory, attention, concentration, and/or global loss of cognitive function. In the present study, we investigated behavioral alterations in sepsis-surviving rats. One hundred and ten male Wistar rats (3-4 months, 250-300 g) were submitted to cecal ligation and puncture (CLP), and 44 were submitted to sham operation. Forty-four rats (40%) survived after CLP, and all sham-operated animals survived and were used as control. Twenty animals of each group were used in the object recognition task (10 in short-term memory and 10 in long-term memory), 12 in the plus-maze test and 12 in the forced swimming test. Ten days after surgery, the animals were submitted individually to an object recognition task, plus-maze and forced swimming tests. A significant impairment of short- and long-term recognition memory was observed in the sepsis group (recognition index 0.75 vs 0.55 and 0.74 vs 0.51 for short- and long-term memory, respectively (P < 0.05). In the elevated plus-maze test no difference was observed between groups in any of the parameters assessed. In addition, sepsis survivors presented an increase in immobility time in the forced swimming test (180 vs 233 s, P < 0.05), suggesting the presence of depressive-like symptoms in these animals after recovery from sepsis. The present results demonstrated that rats surviving exposure to CLP, a classical sepsis model, presented recognition memory impairment and depressive-like symptoms but not anxiety-like behavior.

Desempenho produtivo e atividade microbiana cecal de coelhos alimentados com dietas contendo diferentes níveis de amido

Com o objetivo de avaliar a influência do amido dietético sobre o rendimento produtivo e a atividade microbiana cecal de coelhos em crescimento, quatro dietas experimentais foram elaboradas de forma a conter níveis crescentes de amido (23, 28, 33 e 38% de amido total na base da MS), os quais foram fornecidos a 32 coelhos da raça Nova Zelândia Branco, distribuídos em gaiolas de engorda individuais, seguindo um delineamento em blocos casualizados. Houve efeito linear decrescente para consumo e conversão alimentar, em que valores de 108,67 g/d e 3,216, respectivamente, foram obtidos para os animais alimentados com 38% de amido dietético. No entanto, o ganho de peso e o rendimento de carcaça não foram influenciados pelos tratamentos. Por outro lado, os valores de pH, as concentrações totais e as proporções molares dos ácidos graxos voláteis não foram influenciados pelos tratamentos, porém, para o ácido propiônico, houve efeito linear decrescente, sendo a maior proporção molar (12,06%) verificada no conteúdo cecal de coelhos alimentados com 23% de amido dietético. O milho-grão apresentou maior influência sobre a atividade microbiana cecal que o amido de milho purificado, porém, sem alterar o desempenho e rendimento de carcaça.

Molecular Identification and Characterization of Ileal and Cecal Fungus Communities in Broilers Given Probiotics, Specific Essential Oil Blends, and Under Mixed Eimeria Infection

Broiler digestive tract fungal communities have gained far less scrutiny than that given corresponding bacterial communities. Attention given poultry-associated fungi have focused primarily on feed-associated toxin-producers, yeast, and yeast products. The current project focused on the use of pyrosequencing and denaturing gradient gel electrophoresis (DGGE) to identify and monitor broiler digestive fungal communities. Eight different treatments were included. Four controls were an Uninfected-Unmedicated Control, an Unmedicated-Infected Control, the antibiotic bacitracin methylene disalicylate plus the ionophore monensin as Positive Control, and the ionophore monensin alone as a Negative Control. Four treatments were two probiotics (BC-30 and Calsporin) and two specific essential oil blends (Crina Poultry Plus and Crina Poultry AF). All chickens except the Unmedicated-Uninfected Control were given, at 15 days of age, a standard oral Eimeria inoculum of sporulated oocysts. Ileal and cecal digesta were collected at pre-Eimeria infection at 14 days of age and at 7 days post-Eimeria infection at 22 days of age. Extracted cecal DNA was analyzed by pyrosequencing to examine the impact of diet supplements and Eimeria infection on individual constituents in the fungal community, while DGGE was used to compare more qualitative changes in ileal and cecal communities. Pyrosequencing identified three phyla, seven classes, eight orders, 13 families, 17 genera, and 23 fungal species. Ileal and cecal DGGE patterns showed fungal communities were clustered mainly into pre- and post-infection patterns. Post-infection Unmedicated-Uninfected patterns were clustered with pre-infection groups demonstrating a strong effect of Eimeria infection on digestive fungal populations. These combined techniques offered added versatility towards unraveling the effects of enteropathogen infection and performance enhancing feed additives on broiler digestive microflora.

Uso de microbiota cecal congelada com crioprotetores em pintos infectados experimentalmente com Salmonella enterica sorovar Enteritidis

Pintos de corte com um dia de idade foram tratados com microbiota cecal cultivada em condição de aerobiose, nos tempos de congelamento de 90, 200, 290 e 360 dias, e associada aos crioprotetores sacarose, trealose, dimetilsulfóxido (DMSO) e glicerol. Posteriormente as aves foram desafiadas com Salmonella Enteritidis, visando determinar a eficácia dos tratamentos em relação à quantidade de bactérias viáveis da microbiota que foi maior aos 90 dias (10,58 Log10 UFC/ml), quando as aves foram tratadas com sacarose, e menor aos 290 dias, quando tratadas com glicerol (7,73 Log10 UFC/ml). No tempo zero, todas as aves apresentaram Salmonella (100%) quando tratadas com DMSO e glicerol, com colonização cecal de 4,9 e 5,2 Log10 UFC/g do conteúdo cecal, respectivamente; aos 360 dias nenhuma ave foi infectada, independente do tratamento. A microbiota cecal, independente de tratamento, sempre determinou menor quantidade de S. Enteritidis em qualquer um dos parâmetros pesquisados, quando comparada com a das aves não tratadas. O congelamento em nitrogênio líquido foi eficaz na manutenção da viabilidade da microbiota cecal até 360 dias.

Efeito protetor da microbiota cecal congelada e liofilizada sobre a infecção experimental de frangos de corte por Salmonella enterica sorovar Enteritidis

Estudaram-se os efeitos do tratamento de frangos de corte com microbiota cecal anaeróbia liofilizada (MCL) e congelada (MCC) sobre a infecção do trato digestivo das aves por Salmonella enterica sorovar Enteritidis. Foi usada microbiota intestinal sem prévia identificação bacteriana. A infecção foi persistente, em ordem, no ceco, inglúvio e duodeno. A infecção também foi autolimitante nos grupos tratados e no controle. Não ocorreu diferença entre o grupo-controle positivo e os tratados com MCL ou MCC. Houve redução da colonização do ceco no período de 12 dias após o desafio nos grupos tratados com MCL e MCC, o que não ocorreu no grupo-controle positivo. Não houve variação entre os tratamentos com MCL e MCC quanto às características pesquisadas. A S. Enteritidis reduziu o ganho de peso médio nas aves inoculadas. Os tratamentos com MCL e MCC minimizaram a redução de peso nos grupos infectados.

Efeito da vacina contra coccidiose sobre a colonização de Salmonella enteritidis em frangos de corte tratados com microbiota cecal anaeróbia

Avaliou-se o efeito da vacina contra coccidiose sobre a habilidade da microbiota cecal anaeróbia (MCA), administrada por diferentes vias (aves tratadas), em proteger pintos de corte da colonização por Salmonella enteritidis (Se). Utilizaram-se 120 aves assim divididas: grupo A, pulverização de MCA em aves vacinadas contra coccidiose e desafiadas com Se, grupo B, inoculação endoesofágica de MCA em aves vacinadas e desafiadas, grupo C, MCA na água de bebida de aves vacinadas e desafiadas, grupo D, aves não tratadas, vacinadas e desafiadas, grupo E, aves não tratadas, não vacinadas e desafiadas e, grupo F, aves não tratadas, não vacinadas e não desafiadas (controle negativo). Utilizaram-se como parâmetros a colonização do trato digestivo por Se e sua presença nas fezes, bem como o peso corporal das aves, avaliados aos dois, sete e 12 dias após o desafio. Nas aves tratadas com MCA, especialmente por meio de pulverização e inoculação endoesofágica, a colonização do ceco por Se e sua presença nas fezes foram menores, mostrando que a ação da MCA contra a colonização de ceco e excreção fecal não foi afetada pelo uso da vacina contra coccidiose em pintos de corte e que a associação MCA/vacina contra coccidiose pode ser utilizada sem que haja comprometimento na eficácia da MCA. Nos grupos que não receberam MCA, vacinados ou não contra coccidiose, houve aumento da colonização cecal, bem como excreção fecal da amostra desafio. O ceco foi o local de maior presença e persistência da Se. O resultado de administração de MCA pela água de bebida não foi tão eficiente, mas somente este tratamento resultou em peso corporal das aves significativamente superior ao das aves do grupo não tratado, não vacinado e desafiado, indicando que a presença de salmonelas paratifóides não interfere na produtividade de frangos de corte. Não se observou diferença no peso das aves dos grupos D (vacinados contra coccidiose) e E (não vacinadas) desafiadas com Se, demonstrando que a vacina não influenciou negativamente o ganho de peso das aves desafiadas com Salmonela enteriditis.

Efeito da microbiota cecal e do Lactobacillus salivarius inoculados in ovo em aves desafiadas com Salmonella enterica sorovar Enteritidis

Ovos embrionados provenientes de matrizes pesadas foram inoculados na câmara de ar com microbiota cecal total, microbiota cecal diluída e cultura de Lactobacillus salivarius, no 18º dia de incubação. Dois dias após o nascimento, as aves foram desafiadas com Salmonella enterica sorovar Enteritidis (SE) e, cinco dias após o desafio, avaliou-se a presença da bactéria no fígado e ceco. O efeito de exclusão competitiva, após o desafio com SE, somente foi observado pela ausência da bactéria no fígado das aves tratadas in ovo com L. salivarius. A inoculação in ovo de microbiota cecal indefinida ou diluída não reduziu a colonização de SE no fígado e no ceco das aves, incluindo, neste último, também o tratamento com L. salivarius. Nenhum dos tratamentos in ovo determinou índice de eclodibilidade superior a 65%.

Use of anaerobic cecal microflora, lactose and acetic acid for the protection of broiler chicks against experimental infection with Salmonella Typhimurium and Salmonella enteritidis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)