Doctor of Philosophy / Diagnostic Medicine/Pathobiology / David G. Renter / Shiga toxin-producing Escherichia coli serogroups (O26, O45, O103, O111, O121, O145, and O157; STEC-7) are recognized as major food-borne pathogens with outbreaks, human infections, and occasional deaths associated with the consumption of contaminated foods. Cattle are recognized as the primary reservoir for STEC-7 and shed these bacteria in their feces, which are considered a principal source of contamination of cattle hides and carcasses at harvest. Pre-harvest interventions that effectively reduce fecal shedding of STEC-7 have the potential to reduce the public health concerns and economic impact of these bacteria and enhance food safety. In the research presented in this dissertation, distinct study designs were used to evaluate the impact of commercially available pre-harvest interventions and develop a better understanding of the epidemiology of STEC-7 in commercial feedlot cattle. A randomized pen-level trial indicated that a commercially available vaccine significantly reduced the fecal prevalence of STEC O157 and prevalence of high shedders compared to unvaccinated pens. However, there was no evidence of a direct-fed microbial (DFM) effect on either measure of STEC O157 shedding. In a continuum of the efficacy study, the performance and carcass characteristics associated with these pre-harvest interventions were quantified. Results indicated that feeding the DFM to cattle improved performance, whereas the vaccine negatively impacted performance during the intervention period, though most of these attributes were not reflected at the time the animals were harvested. Later, a cross-sectional observational study was used to determine the regional-, feedlot- and pen-level fecal prevalence of enterohemorrhagic Escherichia coli (EHEC), a subset of STEC, in commercial feedlot cattle. Results indicated that EHEC serogroup O157 was detected more frequently than non-O157 serogroups of EHEC; however, all feedlots had at least one sample positive for both O157 and non-O157 EHEC. Further, risk factors associated with non-O157 serogroups of EHEC were identified; further evaluation of these factors as potential control points may enable the ability to positively impact public health concerns and food safety by reducing the pathogen load prior to harvest. Overall, the research described in this dissertation provides an assessment of pre-harvest interventions and multi-level prevalence estimates of STEC-7 in commercial feedlot operations.

Enterohämorrhagische Escherichia coli (EHEC) gehören zu den wichtigsten der sich in jüngster Zeit verbreitenden Pathogene und verursachen die verschiedensten Durchfallerkrankungen von unblutiger Diarrhö bis zu hämorrhagischer Kolitis, oftmals unter Ausprägung von lebensbedrohlichen extraintestinalen Symptomen wie dem hämolytisch-urämischen Syndrom. Die wichtigsten Virulenzfaktoren dieser Pathogene sind Shiga-Toxine (Stx) und Faktoren, die an der Ausprägung der sog. "attaching and effacing"-Läsionen auf Darmepithelzellen beteiligt sind. Vor allem Kinder und ältere Menschen sind von den Infektionen, die häufig in Form von Ausbrüchen auftreten, betroffen. Die Übertragung erfolgt meist über fäkal kontaminierte Nahrungsmittel. Da die Behandlung von EHEC-Infektionen mit manchen Antibiotika die Entwicklung der extraintestinalen Symptome noch verstärken kann, wäre die Impfung gefährdeter Personen der beste Weg für die Bekämpfung dieser Erreger. Eine weitere Möglichkeit der Prävention wäre die Eradikation dieser Organismen in ihren asymptomatischen Wirten, über die EHEC in die menschliche Nahrungskette gelangen können. Das Ziel der vorliegenden Arbeit war unter anderem die Etablierung der Grundlagen für einen Lebendvakzinstamm zur Prävention von EHEC-Infektionen. Zu diesem Zweck wurden unterschiedliche Strategien mit dem Ziel verfolgt, einen Stx2-produzierenden EHEC-Stamm zu attenuieren. Eine Attenuierungsstrategie für EHEC ist die direkte Ausschaltung von Virulenzfaktor-Strukturgenen wie den Toxingenen. Zu diesem Zweck wurde eine stx2-negative Mutante des EHEC-Stammes O157:H7 86-24 durch eine Deletion im Zentrum des stx2-Genclusters konstruiert, was zur Fusion der 154 N-terminalen Aminosäuren von StxA2 mit den 62 C-terminalen Aminosäuren von StxB2 führte. Die Charakterisierung der Mutante zeigte, daß der Toxin-konvertierende Bakteriophage noch intakt war. Das Fusionsprotein hatte seine zytotoxische Aktivität zwar vollständig verloren, konnte jedoch durch Stx2-spezifisches Schweineantiserum detektiert werden. Daraus wurde geschlossen, daß das mutierte Protein einen Teil seiner antigenen Strukturen behalten hatte und daß es potentiell für die Impfung gegen Stx2-spezifische Schädigungen verwendet werden könnte. Eine weitere Strategie mit dem Ziel der Attenuierung von EHEC-Stämmen war die Deletion von Genen, die in die Regulation von Virulenzfaktoren involviert sind. Auf diese Weise sollte die Expression von Pathogenitätsfaktoren verhindert werden. Als erstes wurde versucht, einen postulierten bakteriophagenkodierten toxinspezifischen Regulator zu identifizieren und zu charaktierisieren, der die Fähigkeit besaß, die Expression eines stx2-spezifischen Reportergens nach der Induktion des Phagen zu steigern. Eine Transposonmutagenese des Stx2-konvertierenden Phagen 933W ergab verschiedene Phagenmutanten mit veränderter Expression des Reportergens nach Induktion des Phagen. Die Expressionsveränderung korrelierte nur bedingt mit der Veränderung der Produktion von Toxin oder Phagenpartikeln. Das Transposon der am stärksten in ihrer Reportergenexpression reduzierten Mutante war im ORF L0065 inseriert, der unmittelbar "upstream" von den Phagengenen int/xis lokalisiert ist. Der klonierte wildtypische ORF war nicht in der Lage, die Transposonmutante in trans zu komplementieren. Daraus wurde geschlossen, daß der Phänotyp der Mutante durch einen polaren Effekt des Transposons auf int/xis bedingt sein könnte, da eine reduzierte Phagengenomexcision eine Verminderung der Phageninduktion verursachen würde, was sich entsprechend auf die Reportergenexpression auswirken könnte. Neely et al. (1998) identifizierten den Phagen-Antiterminator Q als einen möglichen Kandidaten für den postulierten phagenkodierten stx2-Regulator. Eine Deletion dieses zentralen Phagenregulators könnte durch die Störung der regulären Phagenfunktionen zur Attenuierung von EHEC beitragen. Als zweites wurde in einem Projekt von Dr. I. Mühldorfer anhand von recA-negativen Mutanten der EHEC-Stämme O157:H7 86-24 und EDL933 in verschiedenen Mäusemodellen demonstriert, daß die Deletion von recA einen massiven Virulenzverlust und damit eine Attenuierung der Stämme zur Folge hatte. Die dadurch bedingte drastische Reduktion der Toxinproduktion konnte indirekt auf das Fehlen von recA zurückgeführt werden. Im Gegensatz dazu veränderte die Deletion von recA im UPEC-Stamm O6:K15:H31 536 die Virulenz dieses Stammes nicht. Im Rahmen dieser Arbeit erfolgte die Auswertung der Ergebnisse der Virulenztests. Die Deletion von recA ist außerdem eine wichtige Sicherheitsmaßnahme für eine Prävention der Integration von Fremd-DNA in Lebendvakzine und damit für die Verhinderung der Reversion dieser Stämme zur Pathogenität. Als drittes wurden die Auswirkungen der Deletion des Gens leuX, das für die seltenere Leucin-spezifische tRNA5Leu kodiert, auf die Expression von EHEC-Virulenzfaktoren anhand einer leuX-Deletionsmutante des EHEC-Stammes O157:H7 86-24 untersucht. Die Deletion dieser tRNA im UPEC-Stamm 536 führt wegen der dadurch reduzierten Expression verschiedener Virulenzfaktoren zu einer Attenuierung des Stammes. Es wurde gezeigt, daß wie in UPEC auch in EHEC die Produktion von Flagellen und Enterobaktin beeinträchtigt war. Zusätzlich war die Häminverwertung reduziert. Außerdem verminderte die Deletion von leuX die Expression nicht-identifizierter Proteine der äußeren und inneren Membran sowie eines mit Typ 1-Fimbrien-spezifischem Serum kreuzreaktiven Antigens. Im Gegensatz dazu wurden die Stx2-Produktion sowie die in vivo-Virulenz des Stammes in Mäusen nicht beeinflußt. Die Enterohämolyse sowie die Expression von Intimin waren verstärkt. Die Expression der typischen EHEC-Virulenzfaktoren war demnach in der leuX-Mutante nicht reduziert. Der Einfluß von leuX auf die Expression dieser Faktoren war offensichtlich nicht auf eine Translationsreduktion durch die fehlende Bereitstellung der tRNA beschränkt, sondern scheint weitere Mechanismen zu involvieren. Eine wirkliche Attenuierung von EHEC kann durch die Deletion von leuX wahrscheinlich nicht erzielt werden. / Enterohaemorrhagic Escherichia coli (EHEC) are important emerging pathogens responsible for the development of diarrheal diseases, ranging from unbloody diarrhea to hemorrhagic colitis, and of life-threatening extraintestinal complications like the hemolytic-uremic syndrome. The most important virulence factors of this pathogen are the mostly phage-encoded Shiga toxins (Stx) and the factors involved in the development of so-called "attaching and effacing lesions" on gut epithelial cells. Children and the elderly are mainly affected by these infections, which often occur as outbreaks. The infections are predominantly transmitted by fecally contaminated food. The treatment of EHEC infections with some antibiotics may promote the development of extraintestinal complications. Therefore, the best way to combat this infectious agent would be the vaccination of the endangered people. Another way would be the eradication of the organism in its asymptomatic carrier animals involved in the transmission of EHEC into the human food chain. The objective of this thesis was to lay the basis for the development of a life vaccine strain for people or cattle against EHEC infections. Therefore, different strategies aiming at the attenuation of a Stx2-producing EHEC wildtype strain were followed. One strategy for the attenuation of EHEC is the direct knockout of virulence factor structural genes, like the toxin genes. Therefore, a stx2-negative mutant of the EHEC strain O157:H7 86-24 was constructed by deleting the central part of the stx2 gene cluster, leading to the fusion of the 154 N-terminal aminoacids of StxA2 with the 62 C-terminal aminoacids of StxB2. The characterisation of the mutant revealed that the toxin-converting bacteriophage was still intact, but that the fusion protein had completely lost its cytotoxic activity and that it could be detected using Stx2-specific pig antiserum. It has been concluded that the respective mutant protein had kept part of its antigenic structure and that it could potentially be used for vaccination against Stx2-specific damage. Another strategy aiming at the attenuation of EHEC-strains was the deletion of genes involved in the regulation of virulence factors in order to prevent the expression of the respective factors. Firstly, the identification and characterisation of a formerly postulated bacteriophage-encoded toxin-specific regulator which had the capability to induce the expression of a stx2-specific reporter gene after induction of the phage was attempted. A transposon-mutagenesis of the Stx2-converting phage 933W yielded a variety of phage mutants with altered expression of the reporter gene upon phage induction. Toxin production as well as the capability to produce phage particles did not correlate well with the reporter gene phenotypes. The transposon of the mutant with the lowest induction of the reporter gene was inserted into ORF L0065 located immediately upstream of the phage's int/xis genes. The cloned wildtype ORF was not able to trans-complement the transposon mutant. It was concluded that the mutant's phenotype was due to a reduced excision of the phage genome caused by a polar effect of the transposon on int/xis and therefore reduced phage induction leading to reduced reporter gene induction. Neely et al. (1998) identified the phage antiterminator Q as a possible candidate for the postulated phage-encoded stx2 regulator. The specific deletion of this general phage regulator might help to attenuate EHEC by disturbing regular phage functions. Secondly, recA-negative mutants of the EHEC-strains O157:H7 86-24 and EDL933 were examined in different mouse models as part of a project of Dr. I. Muehldorfer. It was demonstrated that the deletion of recA brought about a massive virulence loss due to a drastic reduction of toxin production, which was indirectly caused by the lack of recA. Thus, the deletion of recA attenuates pathogenic EHEC strains in the mouse model. In contrast, the deletion of recA in UPEC strain O6:K15:H31 536 did not alter the virulence of this strain. The analysis of the revealed virulence data was performed as part of this thesis. In addition, the deletion of recA is an important safety measurement for preventing the integration of foreign DNA into attenuated strains and thus, it helps preventing reversion of the vaccine to pathogenicity. Thirdly, the consequences of a deletion of the gene leuX coding for the minor Leucin specific tRNA5Leu on the expression of virulence factors in EHEC were examined by the construction and characterisation of an EHEC O157:H7 86-24 leuX-deletion mutant. In UPEC strain 536, the deletion of this tRNA lead to an attenuation of this strain due to reduced expression of diverse virulence factors. It was demonstrated that in EHEC, like in UPEC, the production of flagella and enterobactin was reduced. In addition, Hemin utilisation was impaired. The deletion of leuX also diminished the expression of various proteins of the outer and inner membrane as well as of an antigen cross-reacting with serum specific for type 1-fimbriae. In contrast, it did not influence the production of Stx2 as well as the in vivo pathogenicity of the strain in mice. Enterohaemolysis and the expression of Intimin were enhanced. Thus, it was demonstrated that the typical EHEC virulence factors were not reduced in the leuX mutant. In addition, it became obvious that the impact of leuX on the expression of the respective genes is not only based on a translational reduction due to a lack of tRNA availability but seems to involve further mechanisms. We concluded that apparently, an attenuation of EHEC is not possible by the deletion of leuX.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2014-09-29T17:42:22Z No. of bitstreams: 3 license_text: 22302 bytes, checksum: 1e0094e9d8adcf16b18effef4ce7ed83 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2014LuisAlbertoPereiraMachado.pdf: 1981082 bytes, checksum: 5d67114fb9c3571e082ce983723758e1 (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2014-10-06T14:03:17Z (GMT) No. of bitstreams: 3 license_text: 22302 bytes, checksum: 1e0094e9d8adcf16b18effef4ce7ed83 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2014LuisAlbertoPereiraMachado.pdf: 1981082 bytes, checksum: 5d67114fb9c3571e082ce983723758e1 (MD5) / Made available in DSpace on 2014-10-06T14:03:17Z (GMT). No. of bitstreams: 3 license_text: 22302 bytes, checksum: 1e0094e9d8adcf16b18effef4ce7ed83 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) 2014LuisAlbertoPereiraMachado.pdf: 1981082 bytes, checksum: 5d67114fb9c3571e082ce983723758e1 (MD5) / A carne suína representa importante fonte de proteína animal para o mundo, porém vem sendo associada a surtos de toxinfecção alimentar. Uma das causas destes surtos é a contaminação por Escherichia coli (E. coli), encontrada no trato intestinal e ambiente dos suínos abatidos para produção de carnes in natura e industrializadas. No contexto de segurança alimentar, os surtos por Escherichia coli produtoras de toxina Shiga (STEC) são os melhores documentados. No mundo, diversos surtos causados por ingestão de alimentos de origem animal já foram documentados, porem no Brasil, existem poucos dados sobre a ocorrência de STEC em eventos de toxinfecção alimentar, bem como a presença nos animais e, consequentemente, na carne suína. O objetivo deste trabalho foi quantificar a contaminação por E. coli de carcaças suínas abatidos em frigoríficos comerciais localizados nos estados da Região Sul do Brasil, e identificar por Reação em Cadeia da Polimerase (PCR) a presença de E. coli produtora das toxinas stx1 e eaeA. Foram realizados swabs de 272 carcaças suínas em abatedouros frigoríficos localizados nos estados do RS, SC e PR. As contaminações por E. coli foram identificadas em 25 carcaças, sendo 20 estabelecimento do Rio Grande do Sul, cinco no do Paraná e nenhuma amostra positiva na coleta em Santa Catarina. Das amostras positivas foram extraídos DNA para genotipagem por PCR. Nenhuma amostra apresentou o gene stx1, porém o gene eaeA foi identificado em 13 amostras, nas diferentes regiões da carcaça. A identificação da incidência dos genes eaeA e stx1 nas carcaças pela técnica de PCR pode ser uma ferramenta útil no rastreamento da contaminação bacteriana ao longo dos processos do abatedouro, podendo auxiliar na redução de casos de toxinfecções alimentares causadas por E. coli e outros microrganismos.

Shigatoxin-producing Escherichia coli (STEC) can cause infections in humans which can beserious and sometimes fatal. There is a great need for methods that are able to detect differentserogroups of STEC. In this project, conventional and real-time PCR assays for detection ofSTEC O104:H4 and O121, as recommended by the European Union Reference Laboratory(EU-RL) for STEC, were validated. The specificity, limit of detection, repeatability,efficiency and robustness were determined for three real-time PCR assays. The validationshowed that the real-time PCR reactions were specific and sensitive although some additionaltests are required.

Freie Universiẗat, Diss., 2004--Berlin. / Dateiformat: zip, Dateien im PDF-Format.- Erscheinungsjahr an der Haupttitelstelle: 2004.

Typical Hemolytic Uremic Syndrome (HUS) presents as a complication of infection with Shiga-toxin producing E. coli (STEC). While there are many animal models for infection, few show true signs of HUS. Additionally, these models differ greatly from the clinical presentation that affects small children and elderly populations. Immunohistochemical assays of tissues from a known HUS model may provide insight into molecular changes associated with the condition, particularly as it pertains to clotting factors. In this study, tissue factor (TF) was investigated in the kidneys of non-human primates previously injected with Shiga-Toxin 2 (STX2). The animals’ condition was indicative of HUS through three main clinical signs: thrombocytopenia, hemolytic anemia and decreased kidney function. Tissue factor antigen in the kidneys varies between animals that exhibited HUS when compared to those that had recovered or treated with anti-STX2 antibody. Overall, tissue factor is strongly detected in the renal tubules of those afflicted with HUS; tissue factor was not strongly expressed in the glomerular epithelial space, as it was in recovered, clinically healthy animals. This suggests a change throughout the time course of disease and recovery. Investigating tissue factor’s role, if any, in the pathology of the disease could lead to new therapeutics. Although many types of treatments have been suggested and tried, the primary clinical procedure is to administer fluids and allow symptoms to subside. With increasing knowledge about HUS through studies like these, we can hope to gain insight into potent therapeutics and therefore, save lives associated with typical HUS.

Les Escherichia coli producteurs de Shiga-toxines (STEC) sont des bactéries pathogènes originaires du tractus digestif des ruminants qui peuvent contaminer certains aliments. Une fois ingérés, ils peuvent provoquer des pathologies graves. Le comportement des STEC n’appartenant pas au sérotype O157:H7 dans les fromages était mal connu. Par ailleurs, en dépit d’une forte prévalence des STEC dans les fromages, cet aliment est moins impliqué dans les épidémies que la viande. C’est pourquoi nous avons étudié dans un premier temps le devenir de ces STEC au cours de la fabrication de 5 schémas technologiques de fromages différents. Dans un deuxième temps, nous avons analysé le comportement et la survie de STEC ayant survécu à la fabrication d’un fromage au cours d’une digestion artificielle. Nos résultats ont montré que certaines technologies sont favorables à la croissance des STEC. Seul un affinage de plus de 60 jours a permis une réduction du taux des STEC. Les étapes de cuisson du caillé ou de coagulation longue de certaines technologies semblent inhiber la croissance des STEC. De plus, la croissance et la survie observées pour les souches E. coli O157:H7 étaient plus faibles que pour les souches E. coli non-O157:H7. L’étude de la survie des souches STEC au cours de la digestion artificielle a montré une différence de comportement entre les 2 souches STEC testées : la souche E. coli O157:H7 a survécu à la digestion tandis que celle de sérotype O26:H11 s’est fortement développée en fin de digestion. / Shiga-toxin producing Escherichia coli (STEC) are an important cause of foodborne illness. They are originated from the digestive tract of the ruminants and can contaminate certain food. Once ingested, they can be responsible for a variety of clinical outcomes. To date, most authors have focused their work on the behavior of E. coli O157:H7 during cheese manufacturing: little is known about the behavior of non-O157:H7 STEC. Furthermore, the presence of STEC strains has been found in raw milk cheeses but dairy products remain less implicated in outbreaks than meat. Consequently, we chose to study the behavior of O157:H7 and non-O157:H7 STEC strains during the manufacturing of 5 different cheese technologies. Then, secondly, it appeared interesting to evaluate the survival of STEC isolated from cheeses during artificial digestion. Our results showed that certain technologies are favorable to the growth of the STEC. Only a long ripening step (>60 days) could then allow a reduction of the STEC rate. In contrast, the cooking step of the curd or the long coagulation of certain technologies inhibited their growth. Our results also showed that the growth and the survival of the E. coli O157:H7 strains were lower than those of the non-O157:H7 E. coli strains. The study of the survival of STEC strains during the artificial digestion highlighted an important difference of behavior for the 2 strains tested: the E. coli O157:H7 strain was able only to survive whereas the E. coli O26:H11 strain knew an important growth at the end of digestion.

Les Escherichia coli producteurs de Shiga-Toxines (STEC) sont responsables de maladies humaines sévères. Les ruminants sont considérés comme étant leur principal réservoir. La dissémination des STEC au sein des élevages est liée en partie à l’alimentation des animaux et donc potentiellement à l’ingestion d’ensilages contaminés. Les bactéries lactiques peuvent être employées comme agents technologiques ou dans des stratégies de bio-protection. Sur le plan de l’ensilage, elles jouent un rôle de préservation mais peuvent également représenter une barrière à la survie de pathogènes comme les STEC. Ce travail a permis de sélectionner des bactéries lactiques inhibitrices de la croissance de divers sérogroupes de STEC. Les études de compétitions ont mis en évidence un phénomène bactéricide sur certaines souches, dont le mécanisme reste encore non élucidé. Le potentiel inhibiteur des bactéries lactiques sélectionnées a été testé indépendamment dans des ensilages de maïs contaminés à différentes étapes de leur réalisation : à la mise en silos, à l’ouverture ou après une période d’exposition aérobie. En cas de contamination à la mise en silos, les souches de STEC testées n’ont pas survécu dans des ensilages correctement menés. Une souche de Ln. mesenteroides a permis de limiter la survie des souches de STEC dans les ensilages contaminés à l’ouverture. Cependant, après 144h d’aération, aucun additif n’a montré d’effet protecteur avéré. Le contrôle de l’alimentation animale afin de limiter l’entrée des STEC dans le cycle épidémiologique pourrait donc passer par l’emploi de bactéries lactiques ; sans négliger cependant les Bonnes Pratiques nécessaires à la réalisation de l’ensilage. / Shiga-toxin producing Escherichia coli (STEC) are responsible for severe human diseases. Cattle are considered as the main reservoir of this pathogen. STEC dissemination in farm environment is linked to cattle feed and potentially to ingestion of contaminated silage. Lactic Acid Bacteria (LAB) could be employed as starters in fermentations or in strategies of bioprotection. In silage, LAB play a preservative role and could also represent a barrier for the survival of pathogenic bacteria such as STEC. The selection of LAB strains, able to inhibit the growth of several serogroups of STEC strains, was performed in this study. Competitions assays have shown a bactericidal effect on some STEC strains, but reasons of this phenomenon remain unclear. Inhibiting potential of the selected LAB strains was tested independently in corn silages contaminated at different steps of their realizations : at ensiling, at opening or after aerobic exposure. In case of contamination at ensiling, STEC strains tested did not survive in well-made silages. A Ln. mesenteroides strain allowed the limitation of the STEC strains survival in silage contaminated at opening. However, after 144 h of aerobic exposure, no inoculant showed any protective effect. Control of cattle feed, in order to limit STEC entry in their epidemiological cycle, could be reached through LAB utilization ; however, Good Manufacturing Practices involved in silage making should not be omitted.

Orientador: Roberto de Oliveira Roça / Resumo: O objetivo deste estudo foi determinar a excreção de patógenos e inocuidade da carcaça de bovinos alimentados com diferentes níveis de silagem inoculada de grãos úmidos de destilaria desengordurados (WDG). Um total de 100 bovinos machos não castrados, 50% Angus e 50% Nelore, foram divididos aleatoriamente entre quatro dietas (N = 25) compostas por diferentes níveis de silagem de WDG (0, 15, 30 e 45% da matéria seca dietética). Amostras de fezes foram colhidas por meio de suabe da junção reto anal, 15 dias antes do abate, para determinar as ocorrências e quantificação de Escherichia coli produtora de toxina Shiga (STEC), E. coli enteropatogênica (EPEC) e Salmonella spp. por meio da técnica qPCR. Também foram colhidas, 52 dias antes do abate, amostras de fezes de cada animal do piso do curral, logo após defecação, as quais foram submetidas a análises físico-químicas. Logo após o abate, a ocorrência e a contagem de indicadores higiênicos e sanitários, E. coli não patogênica, coliformes totais e bactérias aeróbias mesófilas, assim como, a ocorrência de STEC, EPEC e Salmonella spp., foram determinados a partir de amostras colhidas por meio de esponja da superfície das carcaças das regiões do coxão, flanco, peito e pescoço. Os resultados quantitativos foram submetidos a análises de variância e os dados binários foram submetidos a análises logísticas com razão de chances. Todas as análises estatísticas foram realizadas no software estatístico SAS 9.4 considerando um nível de signifi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to determine the pathogen excretion and carcass safety of cattle fed different levels of inoculated silage from degreased wet distillery grains (WDG). A total of 100 male, 50% Angus and 50% Nelore male bulls were randomly divided into four diets (N = 25) composed of different levels of WDG silage (0, 15, 30 and 45% of dietary dry matter) . Stool specimens were collected by rectal anal junction swab 15 days prior to slaughter to determine the occurrences and quantification of Shiga toxin-producing Escherichia coli (STEC), E. coli enteropathogenic (EPEC) and Salmonella spp. by the qPCR technique. Samples of faeces from each animal on the corral floor were also collected, 52 days before slaughter, immediately after defecation, which were submitted to physical-chemical analysis. Immediately after slaughtering, the occurrence and counting of hygienic and sanitary indicators, non-pathogenic E. coli, total coliforms, and mesophilic aerobic bacteria, as well as the occurrence of STEC, EPEC and Salmonella spp., were determined from samples collected by medium of the surface of the carcasses of the regions of the tail, flank, chest and neck. The quantitative results were submitted to analysis of variance and the binary data were submitted to logistic analyzes with odds ratio. All statistical analyzes were performed in SAS 9.4 statistical software considering a significance level of 5%. The results showed that there was no difference between treatments fo... (Complete abstract click electronic access below) / Doutor

Escherichia coli produtora de toxina Shiga (STEC) está entre os patógenos envolvidos em surtos de doenças transmitidas por alimentos devido ao consumo de vegetais. No entanto, até agora, os relatos sobre a presença de STEC em vegetais no Brasil são escassos. Esse microrganismo é veiculado por alimentos, contaminados direta ou indiretamente por fezes animais, sendo responsável por um amplo espectro de doenças que compreende desde diarréia leve que pode evoluir para colite hemorrágica (CH), até síndrome hemolítico-urêmica (SHU) e púrpura trombocitopênica trombótica (PTT). O presente estudo teve como objetivo investigar a presença de STEC em vegetais orgânicos cultivados na região metropolitana da cidade de São Paulo, Brasil, caracterizando os fatores de virulência stx1, stx2, eae e ehx, bem como o sorotipo. Um total de 200 amostras de vegetais orgânicos (folhas verdes), obtido a partir de três produtores foi analisado quanto à presença de cepas de STEC. Caldo triptona de soja (TSB) suplementado com vancomicina (8 mg / L), cefixima (50 µg / L) e telurito de potássio (2,5 mg / L) foi utilizado na etapa de pré-enriquecimento, com incubação a 37ºC / 24 h, seguido por semeadura em MacConkey Sorbitol (SMAC) e CHROMagar STEC (CHROM). Após incubação a 37ºC / 24 h, as colônias suspeitas foram confirmadas por testes bioquímicos e submetidas a PCR objetivando a detecção dos genes de virulência stx1, stx2, eae, ehx, e os genes fliCH7 e rfbO157. Entre as 200 amostras de vegetais orgânicos analisadas, 30 (15%) foram positivas para E. coli, mas nenhum isolado apresentou os genes de virulência pesquisados. Nossos resultados indicam baixo risco de infecção devido ao consumo destes produtos frescos em São Paulo, Brasil. No entanto, são necessárias mais pesquisas, abrangendo um maior número de amostras e área pesquisada, uma vez que este patógeno já foi encontrado no meio ambiente em estudos anteriores e poucas pesquisas investigaram a presença de STEC em vegetais no Brasil. / Shiga toxin producing Escherichia coli (STEC) strains are among the pathogens involved in foodborne disease outbreaks due to consumption of vegetables. However, reports on the presence of STEC in vegetables in Brazil are lacking. STEC is an important pathogen transmitted by food, directly or indirectly contaminated with animal feces, responsible for a broad spectrum of diseases varying from mild diarrhea to hemorrhagic colitis (HC), syndrome hemolytic uremic (HUS) and thrombotic thrombocytopenic purpura (TTP). This study aimed at investigating the presence of STEC in organic vegetables in the metropolitan region of São Paulo city, Brazil, characterizing the virulence factors stx1, stx2, eae and ehx as well as identifying the serotype. A total of 200 samples of organic vegetables (green leafy), obtained from three organic producers was analyzed for the presence of STEC strains. Tryptic Soy Broth (TSB) supplemented with vancomycin (8mg/L), cefixim (50µg/L) and potassium telurite (2.5mg/L) was used in the pre enrichment step with incubation at 37°C/24 h, followed by plating onto Sorbitol-MacConkey (SMAC) agar and CHROMagar STEC (CHROM). After incubation at 37°C/24 h, presumptive colonies were confirmed by biochemical tests and submitted to PCR targeting the detection of stx1, stx2, eae and ehx virulence genes, as well as fliCH7 and rfbO157. Among the 200 organic vegetable samples analyzed for STEC strains, 30 (15%) were positive for E. coli, but none of them showed the virulence genes studied. These findings indicate low risk of infection due to the consumption of these fresh produce in Sao Paulo, Brazil. However, more research is required, covering a larger number of samples and area, since this pathogen has already been found in the environment in previous studies, and few research investigating the presence of STEC in vegetables has been reported in Brazil.