As toxinas LT são expressas por linhagens de Escherichia coli enterotoxigênicas e possuem marcantes efeitos adjuvantes. No entanto, não se conhece se a exposição prévia à toxina afeta sua atividade adjuvante durante imunizações subsequentes. Assim, o presente estudo avaliou o impacto da imunidade pré-existente nas propriedades inflamatórias e adjuvantes da LT quando inoculada pela via subcutânea. Como antígeno modelo, empregou-se a proteína NS1 do vírus dengue e duas abordagens experimentais distintas: (i) incubação in vitro de LT com anticorpos anti-LT e com o receptor gangliosídio (GM1) antes da administração em camundongos não imunizados em conjunto com a proteína NS1 do vírus dengue; (ii) imunização com NS1 coadministrada à LT em camundongos previamente expostos à LT. Foram avaliados os efeitos inflamatórios locais e os efeitos adjuvantes por meio da resposta imunológica humoral anti-NS1. Nossos resultados indicam que a imunidade prévia contra a LT não afeta seu potencial inflamatório e atividade adjuvante. Além disto, a exposição ao receptor GM1 reduziu as reações inflamatórias locais sem, no entanto, reduzir os efeitos adjuvantes de LT. / LT toxins are expressed by enterotoxigenic Escherichia coli strains and display strong adjuvant effects. Nonetheless, the impact of preexisting immunity on the on LT adjuvant activities is still unknown. Thus, the present study evaluated the impact of pre-existing immunity in the inflammatory and adjuvant properties of LT after subcutaneous administration. the NS1 of dengue virus was employed as a model antigen and two experimental approaches were evaluated: (i) in vitro incubation of LT with LT-specific antibodies and the ganglyoside receptor (GM1) before administration to naïve mice in combination with NS1; (ii) immunization with NS1 co-administered with LT in mice previously exposed to LT. The local inflammatory effects induced by LT were evaluated as wel as the adjuvant effects by means of NS1-specific humoral response. Our results indicate that the LT pre-existing immunity does not affect the inflammatory and adjuvant activities of the toxin. In addition, exposure to GM1 reduced the local inflammatory reactions without affecting the toxin adjuvant effects.

As Proteínas Inativadoras de Ribossomos (RIPs) compreendem uma família de enzimas que inibem a síntese protéica através da depurinação de uma adenina específica do RNA ribossomal. Os membros desta família são classificados como RIPs do tipo I, quando possuem somente o RNA-N-Glicosidase e RIPs do tipo II quando além do domínio glicosidase, também apresentam um domínio de lectina. As RIPs foram mais estudadas em plantas, onde a ricina e a aglutinina, ambas RIP do tipo II de mamona (Ricinus communis), estão entre as primeiras descritas. O presente trabalho teve o objetivo de identificar parálogos da ricina e aglutinina, bem como RIPs do tipo I de mamona e analisar as suas relações filogenéticas. Além disso, validar o uso de 14 potenciais genes de referência para qRT-PCR em cinco estádios do desenvolvimento da semente de mamona. O padrão de expressão gênica por RT-qPCR de todas RIPs de mamona identificadas, também foram analisados nestes mesmos estádios. Um total de 18 genes de RIPs foi identificado em mamona (Rcom RIPs), dos quais 10 foram classificados como do tipo II e 8 do tipo I. As topologias das árvores filogenéticas sugerem que as Rcom RIPs foram originadas a partir de múltiplos eventos de duplicação gênica. Dois modelos evolutivos foram propostos para a radiação das Rcom RIPs, baseados em processos de fusão gênica associado ou não a eventos de duplicação parcial. Os genes Act 2/7, EF β, Ubi, TIP e UBC foram os que apresentaram perfil de expressão mais estável e foram selecionados para subsequente normalização dos dados de expressão das Rcom RIPs. Os genes que codificam as Rcom RIPI 3, 4, 5, 7 e 8 e as Rcom RIPII 1, 2, 4, 5, 6 e 8 são transcritos em sementes, sendo que a Rcom RIPII 1 (ricina) e a Rcom RIPII 2 (aglutinina) foram as mais expressas. O presente trabalho apresenta um modelo evolutivo das Rcom RIPs, o qual pode ser extrapolado para outras espécies de plantas. Este trabalho também demonstra o primeiro esforço para a padronização de genes de referência para RT-qPCR em mamona e o primeiro que apresenta a expressão outras Rcom RIPs, além da ricina e aglutinina. / Ribosome inactivating proteins (RIPs) comprise a family of enzymes that inhibit protein synthesis, after depurination of an adenine-specific ribosomal RNA. The members of this family are classified as type I RIPs, which have a RNA-Nglycosidase domain and type II RIPs encompassing a RNA-N-glycosidase and a lectin domain.The RIPs were more studied in plants, where ricin and agglutinin, both type II RIP of castor bean (Ricinus communis), were the first to be described. This work aimed to: 1) identifine paralogous of ricin and agglutinin, as well as the type I RIPs of castor bean; 2) analyze their phylogenetic relationships; 3) validate the use of 14 potential housekeeping genes for qRT-PCR for five developmental stages of R. communis seeds; 4) analyze the pattern of gene expression by RTqPCR of all RIPs castor identified in these same stages. A total of 18 genes that encode RIPs were identified in castor bean (Rcom RIPs), 10 of which were classified as type II and 8 as type I. The phylogenetic trees topologies suggest that Rcom RIPs were originated from multiple events of gene duplications. Two evolutionary models have been proposed for the radiation of Rcom RIPs based on gene fusion processes associated or not to events of partial duplication. The genes Act 2/7, EF β, Ubi, TIP and UBC presented the more stable expression profile and were selected for further RT- qPCR normalization experiments. The Rcom RIPI 3, 4, 5, 7 and 8 and Rcom RIPI 1, 2, 4, 5, 6 and 8 genes are actively transcribed in seeds, whereas the Rcom RIPI 1 (ricin) and Rcom RIPI 2 (agglutinin) were the most expressed. This paper presents an evolutionary model of Rcom RIPs, which can be extrapolated to other plant species. Also, corresponds to the first effort to standardize housekeeping genes for RT-qPCR in castor bean and the first that shows the expression Rcom RIPs, other than ricin and agglutinin.

O aumento da incidência de florações de cianobactérias potencialmente tóxicas nos mananciais de abastecimento, favorecidas pelo elevado aporte de nutrientes nos corpos d\'água, compromete a qualidade da água de consumo e põe em risco a saúde humana e animal, além de elevar os custos do tratamento de água. A pré-cloração, tem se mostrado uma ótima opção tanto na inativação de cianobactérias como na remoção de cianotoxinas dissolvidas. No entanto, sob certas condições, pode causar lise celular e promover a liberação das toxinas no meio. O objetivo deste trabalho foi avaliar em escala laboratorial, o efeito da pré-cloração, utilizando como agente oxidante o hipoclorito de sódio, sobre a integridade celular de uma linhagem tóxica de Microcystis aeruginosa (LTPNA 08), por citometria de fluxo, e sobre a subsequente liberação e degradação das microcistinas (LR e RR) por LC-MS/ MS. Diferentes dosagens de cloro (0,05, 0,5, 1, 1,5, 2, 2,5, 3, 4 e 8 mg.L-1), tempos de contato (0, 15, 30 e 60 minutos) e densidade celular (1x106 células.mL-1 para os ensaios de jarros e 3,5 x106 células.mL-1 para o ensaio de viabilidade celular) foram utilizadas neste estudo. Os resultados obtidos nos ensaios de jarros mostraram remoções de microcistinas acima de 70% após 60 minutos de exposição ao oxidante, com 100% de remoção em doses de 2,5 e 3 mg Cl2.L-1. Valores de CT (concentração x tempo) acima de 40,66 mg.min.L-1 foram necessários para degradar as microcistinas a concentrações abaixo de 1,0 µg.L-1, exigidos pela organização mundial de saúde (WHO) e pela legislação brasileira de potabilidade da água (Portaria MS nº 2914/2011). Não foi possível verificar a lise celular por microscopia óptica, no entanto, na análise por HPLC-DAD verificou-se degradação de mais de 70% da clorofila-a em todas as dosagens testadas, após 60 minutos de exposição, com a completa degradação nas concentrações de 2,5 e 3 mg.L-1 Cl2, indicando dano celular. Nos ensaios por citometria de fluxo, foi verificada a perda da integridade celular com o aumento da dosagem de cloro aplicada, observando-se a permeabilidade celular máxima, sem a desintegração da célula, na concentração de 2,5 mg.L-1 Cl2. Concentrações de 4 e 8 mg.L1 Cl2 promoveram a lise total das células, impossibilitando a permanência do marcador na célula. A perda dos pigmentos clorofila a e ficocianina ocorreram em concentrações de acima de 2,5 e acima de 1,5 mg.L-1 Cl2, respectivamente. O presente trabalho reforçou a eficiência da cloração na degradação das toxinas e os resultados obtidos podem ajudar as autoridades competentes a otimizar as práticas de cloração utilizadas no pré-tratamento da água. / The increased incidence of blooms of potentially toxic cyanobacteria in supply sources, favored by high input of nutrients in water bodies, compromises the quality of drinking water and affect human and animal health, besides increasing water treatment costs. The pre-chlorination, has proved a great choice both in the inactivation of cyanobacterial cells as in removing dissolved cyanotoxins. However, under certain conditions, can cause cell lysis and release toxins. The objective of this study was to evaluate in laboratory scale, the effect of pre-chlorination, using sodium hypochlorite, on cell integrity of toxic Microcystis aeruginosa (LTPNA 08) using flow cytometry, and the subsequent release and degradation of microcystins (LR and RR) by LC-MS / MS. Different chlorine doses (0.05, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 8 mg.L-1), contact times (0, 15, 30 and 60 minutes) and cell density (1x106 células.mL-1 for jar-test and 3.5 x106 células.mL-1 for cell viability assay) were used in this study. The results obtained in the jar- test showed degradations up to 70% after 60 minutes of exposure, with complete degradation at chlorine doses of 2,5 e 3 mg.L-1. Chlorine exposure (CT) values over 40,66 mg.min.L-1 were required for oxidation of microcystin LR and RR to concentrations below the World Health Organization (WHO) and Brazilian legislation for water potability (Portaria MS nº 2914/2011) guideline value of 1µg.L-1. No differences in cell number was observed by microscopy, however, analysis by HPLC-DAD found chlorophyll-a reductions of more than 70% in all dosages tested after 60 minutes exposure, with values below the limit of quantification for concentrations of 2.5 and 3 mg.L-1 Cl2, indicating cell damage. In assays using flow cytometry, loss of cell integrity was observed with increasing chlorine concentration. The maximum cell permeability without cell disintegration was observed at a concentration of 2.5 mg.L-1 Cl2. Concentrations of 4 and 8 mg.L-1 Cl2 lead to complete cell lysis, making impossible the permanence of SYTOX Green in the cell. The loss of pigment chlorophyll a and phycocyanin occurred in concentrations above 2.5 and 1.5 mg.L-1 Cl2, respectively. This study reinforced the efficiency of chlorination in the toxins degradation and the results can help the water authorities to optimize the chlorination practices used in the pretreatment of water.

A distonia é uma síndrome que consiste de contrações musculares involuntárias que resultam em movimentos distorcidos e repetitivos e/ou posturas anormais. O tratamento pode ser por farmacoterapia, com drogas anticolinérgicas ou com a injeção de toxina botulínica no grupo de músculos afetados. O objetivo do trabalho foi avaliar a deglutição nos pacientes com distonia laríngea, antes e após o tratamento com a toxina botulínica. Nossa hipótese foi que a toxina botulínica modificaria a deglutição dos pacientes com distonia laríngea. Foram avaliados 17 indivíduos adultos, acima de 18 anos de idade, com diagnóstico clínico de distonia laríngea antes e após o tratamento com o uso de toxina botulínica do tipo A, e 20 indivíduos adultos saudáveis como controles. Os participantes foram submetidos à anamnese fonoaudiológica e avaliação videofluoroscópica da deglutição. Os pacientes com distonia foram avaliados antes e 30 dias após a injeção de toxina botulínica, guiada por eletromiografia. Na videofluoroscopia foram avaliadas 6 deglutições de 5mL, sendo 3 na consistência líquida (sulfato de bário 100%, e 3 na consistência pastosa (3g do espessante alimentar ThickenUp Clear, em 50 mL de sulfato de bário (100%) oferecidas em uma colher. A ordem das deglutições foi aleatória. Foram estudadas as fases oral e faríngea da deglutição, com registro de 30 quadros por segundo. Os pacientes com distonia laríngea apresentaram aumento de resíduos na região oral e em valécula e maior número de deglutições. Os pacientes apresentaram tempo de trânsito faríngeo (TTF) menor do que os controles (p<0,01), para os bolos nas consistências líquida e pastosa. O TTF foi menor após aplicação do que antes da aplicação da toxina botulínica, quando da deglutição do bolo pastoso. Portanto, concluiu-se que os pacientes com distonia laríngea, comparado a controles, têm trânsito mais rápido pela faringe, aumento de resíduos na região oral e em valécula e maior número de deglutições para o mesmo volume.Trinta dias após a aplicação da toxina botulínica foi observado diminuição da duração do trânsito pela faringe, com o bolo pastoso, e resposta tardia do movimento do osso hióide em relação à chegada do bolo na faringe. / Dystonia is a syndrome consisting of involuntary muscle contractions that result in distorted and repetitive movements and/or abnormal postures. Treatment may be by pharmacotherapy with anticholinergic drugs or with the injection of botulinum toxin in the affected muscle group. The aim of this study was to evaluate swallowing in patients with dystonia before and after treatment with botulinum toxin. Our hypothesis was that botulinum toxin modify the swallowing of patients with spastic dystonia. Seventeen adult subjects over the age of 18 years with clinically diagnosed dystonia were evaluated before and after treatment with botulinum toxin type A and compared to 20 healthy adults as controls. Participants underwent phonologic anamnesis and videofluoroscopy assessment of swallowing. Patients with dystonia were assessed before and 30 days after injection of botulinum toxin, guided by electromyography. In fluoroscopy, 6 swallows were evaluated of 5ml: 3 in a liquid consistency (100% barium sulfate) and 3 in a pasty consistency (3g of food thickener, ThickenUp Clear) in 50 mL of 100% barium sulfate, offered on a spoon. The oral and pharyngeal phases of swallowing were studied from swallows of random order, with registration of 30 frames per second. Patients with dystonia showed an increase of residue in the oral region and vallecula and greater number of multiple swallows. Patients had less pharyngeal transit time (PTT) than controls (p<0.01) for boluses of liquid and pasty consistencies. PTT was lower after the application of botulinum toxin than before with the swallowing of a pasty bolus. It was concluded that patients with dystonia, compared to controls, have more rapid transit through the pharynx, increased residues in the oral region and vallecula and a greater number of swallows for the same volume. Thirty days after the botulinum toxin, it was observed a shorter pharyngeal transit time with paste bolus, and delayed hyoid movement response to bolus presence in pharynx.

The causative agent of the most devastating honeybee disease, American foulbrood (AFB), is the spore-forming bacterium Paenibacillus larvae. To prevent AFB outbreaks beekeepers prophylactically treat their hives with antibiotics even though it decreases the overall health of uninfected hives. A new treatment for AFB is needed due to recent legislation against using antibiotics, antibiotic resistance developing in P. larvae, and the resilience of P. larvae spores. Bacteriophages, or phages, are an attractive alternative to traditional antibiotics because of their specificity and ability to evolve alongside their target bacterium. In this study, two phage cocktails were developed for the treatment of AFB. The first cocktail was comprised of Brevibacillus laterosporus phages. B. laterosporus is a commensal microbe in most honeybee guts. When treated with B. laterosporus phages, B. laterosporus is induced to produce an antimicrobial toxin to which P. larvae is highly sensitive. Treating AFB infected hives with B. laterosporus phages was able to clear active infections at a rate of 75% as opposed to untreated hives that did not recover. However, B. laterosporus phages did not clear latent P. larvae spores and recovered hives relapsed after treatment. The second cocktail was comprised of P. larvae phages and hives treated with the second cocktail recovered at a rate of 100%, protected 100% of at-risk hives, and treated hives did not relapse with AFB suggesting neutralization of P. larvae spores. A P. larvae phage used in the second cocktail was examined to identify any spore-phage interactions. Results from modified plaque assays, fluorescence from FITC-labeled phages bound to spores, and electron microscopy images all confirm that phages bind to P. larvae spores. Phage therapy for the treatment of AFB is an exciting avenue not only as an alternative to chemical antibiotics, but rather a treatment that can neutralize P. larvae spores.

Les venins animaux sont largement distribués à travers le monde, en particulier dans les régions tropicales et subtropicales. Les venins d'animaux sont utilisés comme un mécanisme de défense, d'immobilisation, et de digestion des proies dans la nature. Les venins sont des mélanges complexes des protéines enzymatiques et non enzymatiques avec de spécifiques fonctions physiopathologiques et les peptides des toxines isolés à partir de venins ciblent principalement les canaux ioniques, les récepteurs de la membrane et les composants du système hémostatique avec une affinité élevée. Les venins de serpents ont également été utilisés comme outils médicaux pour des milliers d'années en particulier dans la médecine traditionnelle chinoise. Par conséquent, les venins peuvent être considérés comme des bibliothèques de mini-drogues dans lesquelles chaque médicament est actif sur un plan pharmacologique. Toutefois, moins de 0,01% de ces toxines ont été identifiés et caractérisés. La nouvelle identification de la toxine se déroule généralement à partir d'un test de dépistage, soit in vivo ou sur une cible pharmacologique à intérêt industriel. Dans ce travail, nous criblons pour des composés bioactifs à partir du venin du serpent égyptien noir Walterinnesia aegyptia, qui est capable d'activer la motilité des spermatozoïdes in vitro chez des souris mâles OF1. / Venomous animals are widely distributed throughout the world especially in tropical and subtropical regions. Animal venoms are used as a defense mechanism or to immobilize and digest prey in nature. In fact, venoms are complex mixtures of enzymatic and non- enzymatic proteins components with specific pathophysiological functions. Toxin peptides isolated from animal venoms target mainly the ion channels, membrane receptors and components of the hemostatic system with high affinity. Snake venoms have also been used as medical tools for thousands of years especially in Chinese traditional medicine. Consequently, venoms can be considered as mini-drug libraries in which each drug is pharmacologically active. However, less than 0.01% of these toxins have been identified and characterized. New toxin identification generally proceeds from a screening test, either in vivo or on a pharmacological target of interest to the industry. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia capable to activate sperm motility in vitro from male mice OF1.

The aim of the study in this thesis was to assess the stability of the mycotoxin, cyclopiazonic acid (CPA) in milk and dairy products processed from contaminated milk. A method was developed to detect CPA in milk and milk products using micellar electrokinetic capillary chromatography (MEKC), a technique of capillary electrophoresis (CE), which was rapid and non-labour-intensive. The quantifying efficiency of CE in detecting CPA was compared to Reverse Phase Liquid Chromatography. Heat-stability of CPA in milk was assessed under different conditions. A longer heat treatment of 60 degrees centigrade for 30 minutes led to a 10% decrease in the level of CPA. The results from this thesis demonstrate that CPA in milk at concentrations found in naturally contaminated milk could not be eliminated by the heat-treatment during milk processing, storage, processing and manufacture of dairy products. Occurrence of CPA in cheese curd, butter or cream following manufacture with contaminated milk was demonstrated. CPA is left in milk despite UV-visible radiation treatment with or without hydrogen peroxide and/or riboflavin. Chemical treatment, which is capable of completely eliminating CPA, is prohibited and impractical to use for milk treatment. Stability of CPA in milk and milk products confirms the potential of the toxin to reach consumers of dairy products. / Doctor of Philosophy (PhD)

[Truncared abstract] Introduction: The assessment of any therapy requires not only an understanding of how that therapy works but also how it affects health related quality of life (HRQOL). Botulinum Toxin A(BoNT-A) therapy for upper limb spasticity management in children with hemiplegic cerebral palsy(CP) is currently under trial. Despite its use for over a decade for lower limb spasticity, little is known about the mechanisms involved in improving motor function and the effect on the child and their familys HRQOL. Both central and peripheral mechanisms are hypothesised[1]. Whilst evidence of improved quality of movement and ability to perform tasks is emerging[2-4], this cannot be directly correlated with an improvement in HRQOL. In addition, the traditional method of assessing child HRQOL by parent proxy reports has come under question[5, 6]. The World Health Organisation now recommends the collection of both parent proxy and child self-reports[7]. Aims: 1. Investigate the corticomotor projections to the upper limb in school aged children with hemiplegic CP and the changes that occur with BoNT-A therapy by transcranial magnetic stimulation (TMS). 2. Investigate the effect of upper limb BoNT-A therapy on HRQOL of school aged children with hemiplegic CP by completion of the PedsQL 4.0 Generic Core Scales and 3.0 CP Module. 3. Determine the concordance between Child Self-Report and Parent Proxy Report scores for the PedsQL 4.0 Generic Core Scales and 3.0 CP Module. 4. Determine the concordance between PedsQL scores and function as assessed by the Melbourne Assessment of Unilateral Upper Limb Function (MUUL). Methods: Design: Prospective randomised pilot study. Setting: Department of Paediatric Rehabilitation, Princess Margaret Hospital, and Centre for Neurological and Neuromuscular Disorders, Perth. Participants: 22 school aged children with hemiplegic CP aged 7yr 0mth-13yr 11mth (12 treatment, 10 control). 3 Treatment: One episode BoNT-A injections (dose 1-2U/kg/muscle) into the upper limb for treatment group. The control group received usual care. ... Conclusion: This pilot study provides preliminary evidence of the effects of upper limb BoNT-A therapy at both a central physiological and a broader quality of life level in school aged children with hemiplegic CP. At a central level, corticomotor pathway reorganisation occurs in the setting of BoNT-A. However the reorganisation is not limited to the affected side pathways suggesting a systemic BoNT-A effect or developmental changes. Similarly, in this pilot study, there was no statistically significant effect of upper limb BoNT-A on the childs HRQOL as assessed by the PedsQL although positive trends were observed 4 for a number of physical and psychosocial domains. The collection of both child self-report and parent proxy reports when assessing HRQOL is recommended, and function needs to be assessed independently. Larger studies across the broader CP population, the design of CP specific HRQOL tools appropriate for use in the higher functioning CP cohort, and alternative better tolerated methods of investigating the motor system in children with movement disorders are recommended.

Azaspiracid-1, a novel marine toxin that contains 9 rings and 20 stereogenic centers, has drawn considerable attention from synthetic groups worldwide due to its structural complexity, which includes a unique trioxabisspiroketal fused to a tetrahydrofuran ring (ABCD rings), a piperidine-tetrahydrofuran spiroaminal system fused to a 2,9-dioxabicyclo[3.3.1]nonane system (FGHI rings), a connecting six-membered cyclic hemiketal bridge (E ring) and a ��,��-unsaturated terminal carboxylic acid side chain. Our efforts toward the total synthesis of azaspiracid-1 led to the completion of both C1-C26 northern and C27-C40 southern halves of azaspiracid-1. Herein, our improved and scalable synthetic studies toward the total synthesis of azaspiracid-1 is described. In particular, an improved and scalable synthesis of sulfone 3.6 with a key one-pot ketalization and methylation of ketone 3.22 to methylated hemiketal 3.24 is illustrated. A total 19 mmol of sulfone 3.6 has been prepared by this approach. An improved and scalable synthesis of aldehyde 3.7 utilizing allyl bromide 3.31 to couple with Evans auxiliary 3.33 has been developed. A total of 10 mmol of aldehyde 3.7 has been prepared by this approach. An improved synthesis toward the ABC ring fragment 3.52 with a high yield Julia coupling step is shown. Large scale improved syntheses of the linkage fragment 3.2, the aldehyde fragment 4.9 and the azide fragment 4.10 of the southern portion of (���)-azaspiracid-1 have been described. With an abundant material prepared by this scalable improved approach, we are confident that completing the total synthesis of (���)-azaspiracid-1 will occur in the near future. / Graduation date: 2013

Shiga toxin-producing Escherichia coli (STEC) has been recognized as an important foodborne pathogen. Of this group, O91 is one of the common serogroups frequently isolated from patients and food in some countries, with O91:H21 being previously implicated in hemolytic uremic syndrome (HUS). Cattle are principle reservoirs for STEC, and studies examining STEC shedding in cattle often include experimental inoculation of strains of interest using antibiotic resistance markers for identifiable recovery. However, indigenous fecal microbes exhibiting similar resistance patterns can confound such studies. Such was the case in a study by our group when attempting to characterize shedding patterns of O91:H21 in calves, leading us to seek other, more effective, markers. Among our strategies was the development of a chromosomally integrated bioluminescence marker via transposon mutagenesis using a luxCDABE cassette from Photorhabdus luminescens and a plasmid borne GFP marker via transformation of the pGFP vector. The luxCDABE marker was inserted on host chromosome at a site that was 27 nucleotides before the stop codon of gene yihL and confirmed to have little impact on important virulence genes and growth rate with a very high stability. In contrast, plasmid borne GFP marker showed poor stability without the application of appropriate antibiotic selection pressure. For calves receiving luxCDABE-marked O91:H21, the fecal counts of the organismranged from 1.2 x 10 3 to 1.3 x 10 4CFU/g at two days post inoculation and decreased to 5.8 to 8.7 x 10 2 CFU/g or undetectable level after two weeks.Intestinal contents sampled from various positions at day 14 post inoculation indicated that cecum and descending colon may be the primary localization sites of this O91:H21 strain. Compared to antibiotic resistance markers, the use of bioluminescence markers does not require the restricted pre-inoculation screening of animals. The enumeration of luxCDABE-marked O91:H21 from feces and intestinal contents was easily accomplished and confirmed reliable by M-PCR analysis under the presence of indigenous bacteria which cannot be eliminated by antibiotic-supplemented selective plates. Therefore, the chromosomal integrated luxCDABE marker may be a better model for the study of STEC colonization and shedding in cattle.