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Inhibitors of intracellular trafficking active against plant and bacterial toxins / Les inhibiteurs de trafic intracellulaire actifs contre les toxines plante et bactériennesGupta, Neetu 24 November 2014 (has links)
Les toxines Shiga (Stx) sont produites par Shigella dysenteriae et certaines espèces d’E. coli transmisent aux humains par la consommation d'aliments contaminés et causant des maladies graves. La toxine Stx est libérée par les bactéries dans l'intestin et par la suite, traverse les vaisseaux sanguins en aval pour atteindre leurs principaux organes cibles, notamment les reins. Les dommages causés aux reins peuvent entraîner des complications graves notamment Le syndrome hémolytique urémique (SHU). A ce jour, il n’existe aucun traitement disponible contre le SHU. Les toxines Stx usent du transport rétrograde intracellulaire pour infester les cellules endothéliales rénales et atteindre leur cible cytosolique, l'ARN ribosomal 28S. Via un screening à haut débit, il a été démontré que le composé Rétro-2 bloque le trafic rétrograde de Stx à l'interface Endosome-TGN, sans affecter la morphologie des organites cellulaires et le trafic des protéines endogènes. Au cours de cette thèse, une analyse des relations structure fonction du composé Retro-2 nous a permis d’identifier les régions de l'inhibiteur qui sont critiques pour l'activité de protection. Nous avons identifié un dérivé dihydroquinazolinone nommé Rétro-2.1 qui est à ce jour l'inhibiteur le plus puissant contre les toxines Stx. Afin d’identifier la cible moléculaire de Retro-2.1, nous avons développé des sondes photo-activables bio-actives. En outre, les données de diffraction des rayons X ont révélé que de l'activité antitoxine réside principalement dans l’énantiomère S. (S) -Retro-2.1 est 500 fois plus puissant contre Stx (50 nM) que la molécule initiale. Cette étude peut donner lieu à un nouveau concept thérapeutique ciblant la voie de transport rétrograde de la toxine à l'intérieur de la cellule hôte. Une telle stratégie thérapeutique pourrait donc être étendue à d'autres agents pathogènes qui usent également du trafic rétrograde pour une intoxication des cellules hôtes. Ce nouveau concept thérapeutique qui permet de cibler les cellules hôtes et non l'agent pathogène représente une véritable percée dans la découverte de médicaments à large spectre et réduit le risque de développement d’une résistance chez l’agent pathogène. / Shiga toxins (Stx) are produced by Shigella dysenteriae and certain species of E. coli that can be transmitted to humans primarily through consumption of contaminated foods and may cause severe disease. Stx is released by the bacteria in the intestine and subsequently, could cross the downstream blood vessels to reach their main target organs such as kidney. Damage to the kidney can result in serious life-threatening complication hemolytic uremic syndrome, for which there is no proven safe treatment available other than supportive care. Stx invades renal endothelial cells in a retrograde manner from cell surface to the endoplasmic reticulum in order to gain access to its cytosolic target, 28S rRNA. By using HTS, it was previously demonstrated that the compound Retro-2 blocks retrograde trafficking of Stx at the early endosome-TGN interface, without affecting the morphology of cellular organelles and trafficking of other endogenous proteins. In this work, different regions of the lead inhibitor Retro-2 that are critical for the protective activity have been determined by systematic structure-activity relationship studies. It allowed us to identify a dihydroquinazolinone derivative, named Retro-2.1 that is the most potent inhibitor of Stx to date and also to develop bio-active photo-activatable probes with the aim of identifying the molecular target of Retro-2 derivatives. Further, crystal X-ray diffraction data revealed that the antitoxin activity resides mainly in the S-enantiomer. (S)-Retro-2.1 has displayed 500 fold more potency (50 nM) than parent molecule against Stx cytotoxicity. This study may result in a new therapeutic concept - targeting the retrograde transport route of toxin inside host cell - for the treatment of Stx-producing E. coli infections and could therefore be extended to other pathogens that also traffic via the retrograde transport. Such a new therapeutic concept that target the host cells and not the pathogen itself would represent a real breakthrough in drug discovery leading to broad spectrum drugs.
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Desenvolvimento de uma estratégia vacinal contra a toxina de Shiga de Escherichia coli enterohemorrágica (EHEC) baseada na proteína recombinante Stx2ΔAB incorporada a lipossomas. / Development of a vaccine strategy against Shiga toxin (Stx) of Escherichia coli (EHEC) based on recombinant protein Stx2ΔAB incorporated into liposomes.Jesus, Monica Josiane Rodrigues de 07 February 2017 (has links)
Infecções associadas a cepas da Escherichia coli enterohemorrágica (EHEC), podem causar manifestações clínicas sendo a Síndrome Hemolítica Urêmica (SHU), a complicação mais severa. SHU está relacionada a presença da toxina de Shiga do tipo 2 (Stx2) e até o momento não se dispõe de uma vacina ou tratamentos efetivos para uso em humanos. Assim, este trabalho teve por objetivo desenvolver uma vacina baseada no derivado atóxico contendo a subunidade B e a porção A2 da subunidade A denominado Stx2ΔAB. Após expressão em linhagens de E.coli e tentativas iniciais de purificação, resultaram na formação de agregados proteicos. Ajustes nas condições de cultivo e purificação permitiram obter a proteína na forma de monômero da subunidade B, mas sem a presença da porção A2. O antígeno foi incorporado a lipossomas multilamelares (MLVs), combinados ao lipídio A e administrados por via subcutânea a camundongos. Animais imunizados desenvolveram anticorpos sistêmicos específicos contra Stx2 capazes de neutralizar a toxina in vitro e conferir proteção parcial a animais desafiados com dose letal da toxina. Em conclusão, o trabalho confirmou o potencial vacinal do antígeno e validou a estratégia baseada na incorporação do antígeno às MLVs como estratégia de imunização. / Infections associated with strains of enterohaemorrhagic Escherichia coli (EHEC), can cause clinical manifestations are the hemolytic uremic syndrome (HUS), the most severe complication. HUS is related to the presence of Shiga toxin type 2 (Stx2) and yet do not have a vaccine or effective treatments for use in humans. This work aimed to develop a vaccine based on non-toxic derivative containing the B subunit and the A2 portion of the subunit called Stx2ΔAB. After expression in E. coli strains and initial purification attempts resulted in the formation of protein aggregates. Adjustments in the cultivation and purification conditions have enabled the protein as the monomer subunit B but without the presence of the A2 portion. The antigen was incorporated into multilamellar liposomes (MLVs), the combined lipid A and administered subcutaneously to mice. immunized animals develop systemic antibodies specific against Stx2 able to neutralize toxin in vitro and to confer partial protection when challenged with a lethal dose of toxin. In conclusion, the study confirmed the potential vaccine antigen and validated strategy based on antigen incorporation into MLVs as immunization strategy.
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Molekularbiologische Charakterisierung Shiga Toxin 1-konvertierender Bakteriophagen und des phagenkodierten Typ III Effektorproteins NleA4795Creuzburg, Kristina 08 June 2007 (has links) (PDF)
Shiga Toxin (Stx)-konvertierende Bakteriophagen besitzen eine konservierte lambdo-ide Genomstruktur, weisen aber ein hohes Maß an genetischer Diversität auf. Aus diesem Grund wurden die bereits vorhandenen Sequenzdaten der Stx1-Phagen CP-1639 des Escherichia coli O111:H- Stammes 1639/77 und BP-4795 des E. coli O84:H4 Stammes 4795/97 vervollständigt und analysiert. Im Gegensatz zu dem induzierbaren Bakteriophagen BP-4795 fehlen CP-1639 einige Gene, die für den lytischen Lebenszyklus eines Bakteriophagen notwendig sind. Daher handelt es sich bei CP-1639 um einen kryptischen Prophagen, der nicht mehr in der Lage ist das Wirtschromosom zu verlassen und intakte Phagenpartikel zu bilden. Die Integra-tionsstellen wurden innerhalb des Gens yehV für BP-4795 und ssrA für CP-1639 bestimmt. In unmittelbarer Umgebung des Integrationsortes von CP-1639 befindet sich ein eigenständiges integratives Element. Dieses besteht aus drei offenen Lese-rahmen unbekannter Herkunft, sowie einem Integrasegen und kann auch ohne Assoziation mit Phagen-DNA auftreten. Phagen können zusätzlich zu ihrem Genom Gene bakteriellen Ursprungs tragen. Diese können unter anderem infolge von Transpositionen oder einer unkorrekten Exzision der Phagen-DNA aus dem Wirtschromosom während des lytischen Lebenszyklus in das betreffende Phagengenom eingebaut werden. Eines solches Gen des Bakteriophagen BP-4795 kodiert das Typ III Effektorprotein NleA4795, dessen Funktionalität nach dem C-terminalen Einbau von neun Codons des Hämagglutinin (HA)-Epitopes des humanen Influenzavirus in die Sequenz des Gens nleA4795 überprüft wurde. Dies erfolgte unter Verwendung der Western Blot Analyse durch die Expression dieses Fusionsproteins in dem Wildtyp-Stamm 4795/97 und in der Deletionsmutante 4795escN des Stammes 4795/97. Die Typ III Sekretionssys-tem (T3SS)-inaktive Mutante 4795escN wurde im Verlauf dieser Arbeit hergestellt. Diese Untersuchungen zeigten ebenfalls, dass zur Sekretion von NleA4795 ein in-taktes T3SS notwendig ist. Weiterhin zeigte die Infektion einer HeLa-Zelllinie mit NleA4795-HA exprimierenden Bakterienstämmen und die anschließende Analyse mit Hilfe der Immunfluoreszenz, die Translokation des Proteins in eukaryotische Wirts-zellen. Darüber hinaus deuten die Ergebnisse dieser Untersuchung auf eine Lokali-sation von NleA4795-HA innerhalb des Trans-Golgi Netzwerkes hin. Die Verbreitung des Gens nleA wurde in insgesamt 170 Shiga Toxin-produzierenden E. coli und enteropathogenen E. coli Stämmen untersucht. Dies führte zur Identifika-tion von 14 verschiedenen Varianten des Gens nleA in 149 der überprüften Stämme, wobei mit Ausnahme von zwei Isolaten ebenfalls ein Markergen des T3SS nachge-wiesen werden konnte. Neben drei bereits bekannten Varianten konnten elf neue Varianten identifiziert werden, deren abgeleitete Aminosäuresequenzen sich zu 71% bis 96% glichen. Sequenzunterschiede traten insbesondere aufgrund der Deletion oder Insertion von vier bis 51 Aminosäuren im Mittelteil der potentiellen Proteine auf. Weiterhin deuten die Ergebnisse dieser Untersuchungen eine Assoziation bestimm-ter Varianten des Gens nleA mit spezifischen E. coli Serogruppen an. Southern-Blot Hybridisierungen zeigten, dass etwa ein Viertel der nleA-positiven Stämme zwei Ko-pien dieses Gens in ihrem Genom tragen. Hierbei handelt es sich meist um zwei verschiedene Varianten. In fast allen Fällen kodiert eine dieser Varianten, aufgrund einer Punktmutation oder Insertion eines IS-Elementes, ein verkürztes und vermut-lich nicht funktionelles Protein. Mit Hilfe von Transduktionsexperimenten konnten verschiedene Varianten des Gens nleA im Genom induzierbarer Phagen nachge-wiesen werden, was auf eine Verbreitung des Gens nleA durch den horizontalen Gentransfer hinweist.
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The pathophysiology of renal failure in a shiga toxin plus lipopolysaccharide induced murine model of hemolytic uremic syndromePsotka, Mitchell Adam. January 2008 (has links)
Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.
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The pathophysiology of renal failure in a shiga toxin plus lipopolysaccharide induced murine model of hemolytic uremic syndromePsotka, Mitchell Adam. January 2008 (has links)
Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online as viewed 8/06/2009 through Digital Dissertations.
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Avaliação da exposição do consumidor à Listeria monocytogenes, Salmonella spp., Campylobacter spp. e Escherichia coli produtora de toxina de Shiga em produtos cárneos refrigerados comercializados no município de São Paulo / Assessment of consumer exposure to Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Shiga toxin-producing Escherichia coli in refrigerated meat products at retail in São Paulo municipalityChristiane Asturiano Ristori Costa 30 March 2010 (has links)
As Enfermidades Transmitidas por Alimentos representam um crescente e relevante problema de saúde pública. Além do prejuízo social, a contaminação de alimentos com microrganismos patogênicos gera um enorme prejuízo econômico. Técnicas de Análise de Risco permitem mensurar de forma mais adequada o impacto dos microrganismos contaminantes de alimentos na saúde da população. Uma Análise de Riscos, associada a uma combinação patógeno-alimento, envolve três passos: avaliação do risco, gestão do risco e comunicação do risco. Uma das etapas da avaliação do risco é a avaliação da exposição, baseada em dados sobre freqüência e nível de contaminação dos alimentos pelo patógeno avaliado no alimento em questão, o nível atingido pelo patógeno no momento do consumo e os padrões de consumo. Os produtos cárneos são os principais alimentos responsáveis pela veiculação de patógenos ao homem e os microrganismos de maior relevância nestes produtos são Listeria monocytogenes, Salmonella spp., Campylobacter spp. e Escherichia coli produtora de toxina de Shiga. O objetivo do presente estudo foi levantar informações qualitativas e quantitativas desses quatro patógenos em produtos cárneos (salsicha bovina, lingüiça suína, carne bovina moída e coxa de frango) comercializados no município de São Paulo, de forma a contribuir com dados para futuras avaliações de risco em relação a estes microrganismos nestes produtos. Das 552 amostras de produtos cárneos analisadas, L. monocytogenes foi o patógeno isolado com maior freqüência, sendo detectado em 48,7% das amostras, seguido por Campylobacter spp. em 6,0% e Salmonella spp. em 5,8%. E. coli produtora de toxina de Shiga não foi detectada em nenhuma das amostras estudadas. Listeria monocytogenes foi detectada em todos os tipos de produtos cárneos estudados, com freqüências mais elevadas nas amostras de carne bovina moída (59,4%), seguido de coxa de frango (58,0%), lingüiça suína (39,8%) e salsicha bovina (37,7%). Na maioria das amostras (94,4%), as contagens de L. monocytogenes foram inferiores a 102 UFC/g. As cepas de L. monocytogenes apresentaram ampla distribuição, sendo detectados os quatro grupos de sorotipos: 28,7% pertenceram ao Grupo 1 (sorotipos 1/2a e 3a), 21,0% ao Grupo 2 (sorotipos 1/2c e 3c), 17,0% ao Grupo 3 (sorotipos 1/2b, 3b e 7) e 13,8% ao Grupo 4 (sorotipos 4b, 4d e 4e). Salmonella spp. foi detectada em 32 amostras, sendo 20 (14,5%) de lingüiça e 12 (10,6%) de coxa de frango. As contagens foram baixas, variando de 3,0 a 9,3x10 NMP/g e os sorovares mais freqüentemente isolados foram S. Typhimurium (28,1%), S. Enteritidis (12,5%), S. Derby (12,5%) e S. I 4,[5],12:i:- (12,5%). Campylobacter spp. foi detectado em 33 amostras (6,0%), sendo 27 de coxa de frango (19,6%) e seis amostras de carne moída (4,3%). A presença de L. monocytogenes, Salmonella spp. e Campylobacter spp. nos produtos cárneos analisados representa um risco à saúde da população. O consumo destes produtos quando submetidos à cocção inadequada e/ou a contaminação cruzada com outros alimentos pode levar a ocorrência de Enfermidades Transmitidas por Alimentos. / Foodborne Diseases represent an increasingly important public health problem. Besides the social losses, contamination of food with pathogenic microorganisms generates an enormous economic damage. A more accurate measurement of the impact of microorganisms in food health can be achieved using Risk Analysis techniques. A risk analysis is composed by three elements: risk assessment, risk management and risk communication. One of the four steps of a risk assessment is the exposure assessment, based on data on frequency and level of contamination of a food by the pathogen under evaluation, levels of the pathogen in the food at the time of consumption and consumption patterns. Meat products are the main vehicles of pathogens to humans, where Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Shiga toxin-producing Escherichia coli are the most relevant pathogens. The aim of this study was to obtain qualitative and quantitative information on these four pathogens in four types of meat products (beef sausage, pork sausage, ground beef and chicken leg) marketed in the city of Sao Paulo in order to contribute with data for future risk assessments for these microorganisms in these products. L. monocytogenes is the most frequent pathogen in the 552 samples of meat products analyzed, being detected in 48.7% of the samples, followed by Campylobacter spp. 6.0% and Salmonella spp. 5.8%. Shiga toxin-producing E. coli was not detected in any sample. L. monocytogenes was detected in all types of meat products, with highest frequency in ground beef (59.4%), followed by chicken leg (58.0%), pork sausage (39.8%) and beef sausage (37.7%). In most samples (94.4%), the counts of L. monocytogenes were below 102 CFU/g. L. monocytogenes strains were widely distributed in the four groups of serotypes: 28.7% belonged to Group 1 (serotypes 1/2a and 3a), 21% to Group 2 (serotypes 1/2c and 3c), 17% to Group 3 (serotypes 1/2b, 3b and 7) and 13.8% to Group 4 (serotypes 4b, 4d and 4e). Salmonella spp. was detected in 32 samples, being 20 (14.5%) of pork sausage and 12 (10.6%) of chicken leg. The counts were low, ranging from 3.0 to 9.3 x 10 MPN/g and the most frequent serovars were S. Typhimurium (28.1%), S. Enteritidis (12.5%), S. Derby (12.5%) and S. I 4, [5], 12: i: - (12.5%). Campylobacter spp. was detected in 33 samples (6.0%), being 27 of chicken leg (19.6%) and six samples of ground beef (4.3%). The presence of L. monocytogenes, Salmonella spp. and Campylobacter spp. in the tested meat products represent a risk to health. The consumption of inadequately cooked products and/or subjected to cross-contamination with other foods may lead to occurrence of foodborne diseases.
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Molekularbiologische Charakterisierung Shiga Toxin 1-konvertierender Bakteriophagen und des phagenkodierten Typ III Effektorproteins NleA4795Creuzburg, Kristina 24 May 2007 (has links)
Shiga Toxin (Stx)-konvertierende Bakteriophagen besitzen eine konservierte lambdo-ide Genomstruktur, weisen aber ein hohes Maß an genetischer Diversität auf. Aus diesem Grund wurden die bereits vorhandenen Sequenzdaten der Stx1-Phagen CP-1639 des Escherichia coli O111:H- Stammes 1639/77 und BP-4795 des E. coli O84:H4 Stammes 4795/97 vervollständigt und analysiert. Im Gegensatz zu dem induzierbaren Bakteriophagen BP-4795 fehlen CP-1639 einige Gene, die für den lytischen Lebenszyklus eines Bakteriophagen notwendig sind. Daher handelt es sich bei CP-1639 um einen kryptischen Prophagen, der nicht mehr in der Lage ist das Wirtschromosom zu verlassen und intakte Phagenpartikel zu bilden. Die Integra-tionsstellen wurden innerhalb des Gens yehV für BP-4795 und ssrA für CP-1639 bestimmt. In unmittelbarer Umgebung des Integrationsortes von CP-1639 befindet sich ein eigenständiges integratives Element. Dieses besteht aus drei offenen Lese-rahmen unbekannter Herkunft, sowie einem Integrasegen und kann auch ohne Assoziation mit Phagen-DNA auftreten. Phagen können zusätzlich zu ihrem Genom Gene bakteriellen Ursprungs tragen. Diese können unter anderem infolge von Transpositionen oder einer unkorrekten Exzision der Phagen-DNA aus dem Wirtschromosom während des lytischen Lebenszyklus in das betreffende Phagengenom eingebaut werden. Eines solches Gen des Bakteriophagen BP-4795 kodiert das Typ III Effektorprotein NleA4795, dessen Funktionalität nach dem C-terminalen Einbau von neun Codons des Hämagglutinin (HA)-Epitopes des humanen Influenzavirus in die Sequenz des Gens nleA4795 überprüft wurde. Dies erfolgte unter Verwendung der Western Blot Analyse durch die Expression dieses Fusionsproteins in dem Wildtyp-Stamm 4795/97 und in der Deletionsmutante 4795escN des Stammes 4795/97. Die Typ III Sekretionssys-tem (T3SS)-inaktive Mutante 4795escN wurde im Verlauf dieser Arbeit hergestellt. Diese Untersuchungen zeigten ebenfalls, dass zur Sekretion von NleA4795 ein in-taktes T3SS notwendig ist. Weiterhin zeigte die Infektion einer HeLa-Zelllinie mit NleA4795-HA exprimierenden Bakterienstämmen und die anschließende Analyse mit Hilfe der Immunfluoreszenz, die Translokation des Proteins in eukaryotische Wirts-zellen. Darüber hinaus deuten die Ergebnisse dieser Untersuchung auf eine Lokali-sation von NleA4795-HA innerhalb des Trans-Golgi Netzwerkes hin. Die Verbreitung des Gens nleA wurde in insgesamt 170 Shiga Toxin-produzierenden E. coli und enteropathogenen E. coli Stämmen untersucht. Dies führte zur Identifika-tion von 14 verschiedenen Varianten des Gens nleA in 149 der überprüften Stämme, wobei mit Ausnahme von zwei Isolaten ebenfalls ein Markergen des T3SS nachge-wiesen werden konnte. Neben drei bereits bekannten Varianten konnten elf neue Varianten identifiziert werden, deren abgeleitete Aminosäuresequenzen sich zu 71% bis 96% glichen. Sequenzunterschiede traten insbesondere aufgrund der Deletion oder Insertion von vier bis 51 Aminosäuren im Mittelteil der potentiellen Proteine auf. Weiterhin deuten die Ergebnisse dieser Untersuchungen eine Assoziation bestimm-ter Varianten des Gens nleA mit spezifischen E. coli Serogruppen an. Southern-Blot Hybridisierungen zeigten, dass etwa ein Viertel der nleA-positiven Stämme zwei Ko-pien dieses Gens in ihrem Genom tragen. Hierbei handelt es sich meist um zwei verschiedene Varianten. In fast allen Fällen kodiert eine dieser Varianten, aufgrund einer Punktmutation oder Insertion eines IS-Elementes, ein verkürztes und vermut-lich nicht funktionelles Protein. Mit Hilfe von Transduktionsexperimenten konnten verschiedene Varianten des Gens nleA im Genom induzierbarer Phagen nachge-wiesen werden, was auf eine Verbreitung des Gens nleA durch den horizontalen Gentransfer hinweist.
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Escherichia coli O157: detection and quantification in cattle feces by quantitative PCR, conventional PCR, and culture methodsNoll, Lance January 1900 (has links)
Master of Science / Department of Diagnostic Medicine/Pathobiology / T. G. Nagaraja / Shiga toxin-producing E. coli O157 is a major foodborne pathogen. The organism colonizes the hindgut of cattle and is shed in the feces, which serves as a source of contamination of food. Generally, cattle shed E. coli O157 at low concentrations (≤ 10[superscript]2 CFU/g), but a subset of cattle, known as “super-shedders”, shed high concentrations (>10[superscript]3 CFU/g) and are responsible for increased transmission between animals and subsequent hide and carcass contamination. Therefore, concentration data are an important component of quantitative microbial risk assessment. A four-plex quantitative PCR (mqPCR) targeting rfbE[subscript]O157, stx1, stx2 and eae was developed and validated to detect and quantify E. coli O157 in cattle feces. Additionally, the applicability of the assay to detect E. coli O157 was compared to conventional PCR (cPCR) targeting the same four genes, and a culture method. Specificity of the assay to differentially detect the four genes was confirmed. In cattle feces spiked with pure cultures, detection limits were 2.8 x 10[superscript]4 and 2.8 x 10[superscript]0 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. Of the 100 samples that were randomly picked from the 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from the 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar’s chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. Applicability of the assay to quantify E. coli O157 was determined with feedlot cattle fecal samples (n=576) and compared to spiral plate method. Fecal samples that were quantifiable for O157 by mqPCR (62/576; 10.8%) were at concentrations of ≥ 10[superscript]4 CFU/g of feces. Only 4.5% (26/576) of samples were positive by spiral plate method, with the majority (17/26; 65.4%) at below 10[superscript]3 CFU/g. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect and quantify E. coli O157 in cattle feces.
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Identification des gènes de Escherichia coli entérohémorragique exprimés pendant l'infection de macrophages humainsPoirier, Katherine January 2007 (has links)
Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.
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Defining the impact of colonisation with Shiga toxin positive E. coli O157 on adaptive immunity in cattleBeckett, Amy Elizabeth January 2018 (has links)
Shiga producing E. coli (STEC) O157 is a zoonotic pathogen. In humans STEC O157 causes bloody diarrhoea and potentially fatal renal failure. Cattle are the major reservoir, where bacteria are limited to the intestinal tract and do not cause clinical signs of disease. Previous studies indicate that shiga toxins produced by STEC O157, suppress STEC-specific cellular immune responses in vivo. This study aimed to initially examine the humoral immune response in cattle following natural challenge and the effects of a toxoid vaccination on this humoral STEC specific-immune response. We determined a statistically significant suppression in Tir specific IgA in STEC O157 positive cattle compared to O157 negative cattle but not in super shedding cattle. Following toxoid vaccination we determined a significant increase in flagellin specific IgG1 antibody levels in toxoid vaccinated animals despite lower numbers of positive faecal samples compared to placebo vaccinated controls. These results suggest that shiga toxins produced by STEC O157 are actively suppressing the STEC specific immune response in natural colonisation. To clarify this suppression further calves were orally challenged with STEC O157 (either a PT21/28 Stx2c+, PT32 Stx2c+ or PT21/28 Stx2a+Stx2c+ strain) and their STEC specific immune responses monitored. STEC specific systemic antibody responses were variable and weak in some cases. STEC specific local antibody responses were only significantly increased following challenge with the PT21/28 Stx2a+Stx2c+ challenge. Transcripts for genes associated with immune responses, and in particular B cell activation, at the terminal rectum were analysed by reverse transcriptase quantitative PCR. Suppression of IL2RA transcripts was observed in calves challenged with PT21/28 Stx2a+Stx2c+ compared to control calves but not with the other two STEC O157 strains tested. This study also aimed to determine the effects of cattle colonisation with STEC O157 on the immune response to a non-bacterial T-cell dependent antigen, ovalbumin (OVA). Cattle were orally challenged with either a PT21/28 Stx2c+, PT32 Stx2c+ or PT21/28 Stx2a+Stx2c+ strain or unchallenged. Calves were subcutaneously immunised with OVA five days post challenge, on two separate occasions with a two week interval. Lymphocytes from lymph nodes local to the immunisation site demonstrated significantly increased OVA-specific proliferation and OVA-specific activation of CD4+ and CD8+ cells in calves that were challenged with the PT21/28 Stx2c+ strain (but not with the other two challenge strains), compared to unchallenged controls. These results indicate that colonisation with STEC O157 can alter local adaptive immune responses to non-bacterial antigens in a strain dependent manner, unexpectedly enhancing the immune response rather than suppressing it. Circulating T cell responses were unaffected. In conclusion this study provides some further evidence of adaption of the host immune response by STEC O157, which is strain dependent, and variable. It seems unlikely from the data in this study that STEC O157 colonisation is having a major impact on the responses of cattle to other vaccines or infections in the field.
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