Dissertations / Theses on the topic 'Eye – Microbiology'

High-pressure needleless injection (HPNI) is a process where small-diameter, high-velocity burst of liquid, penetrate foods at pressures ≤ 10,000 psi. The potential of HPNI as an enhancing technique for meat was studied. In study 1, HPNI translocated surface E. coli O157 into the interior of beef eye-of-round subprimals with an incidence of 40 (±7), 25 (±8), and 25 (±8)% for meat that had been surface-inoculated with a four-strain cocktail at 0.5, 1, and 2 log10 CFU/cm2, respectively. Run-off water contained 2, 2, and 3 log10 CFU/ml and was used for HPNI of additional subprimals, which resulted in a cross-contamination incidence of 83 (±4), 60 (±15), and 37 (±6) %, respectively. Incidence of translocation and cross-contamination was similar at all sampled levels below the inoculated surface. Study 1 results indicate that surface microflora will be translocated from the surface into the interior of HPNI-treated beef by the injection fluid and by cross-contamination with recycled fluid. In study 2, E. coli was undetected in cooked steaks (63˚C internal) cut from subprimals inoculated with 2 log10 CFU/cm2 and HPNI processed (study 1). Although cooking reduced E. coli counts, determination of complete kill was not possible because the detection limit for bacterial recovery was about 1 log10 CFU/g. Steaks cut from HPNI-processed subprimals took longer (p <0.05) to reach 63˚C with grilling or broiling, compared to control steaks, possibly due to increased moisture in enhanced steaks. In study 3, sensory acceptance of steaks was evaluated by a consumer panel. Appearance, flavor, and overall acceptance were similar among the untreated control, HPNI steaks, blade tenderized steaks (BT steaks), and steaks cut from subprimals that had been needle-injected with 0.35% (wt/vol) sodium tripolyphosphate using needle injection (NI-subprimal steaks) or HPNI (HPNI-subprimal steaks). Texture of BT steaks (6.5±1.9) was more liked than control steaks (5.8±1.8), while texture was similar for all other comparisons. Conversely, Warner-Bratzler shear force was NI-subprimal steaks < control < HPNI steaks = HPNI-subprimal steaks = BT steaks. Lack of correspondence between texture acceptance data and WBSF suggests that sensory scores were influenced by factors other than the force required for mechanical shear.

Title: Treatment of CMV Vitritis in a Preterm Newborn Author’s Section: Remil Simon1, Darshan Shah1, Peter Blosser1, Demetrio Macariola1, Jeffrey Carlsen2 1.Department of Pediatrics, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 2.Johnson City Eye Clinic, Johnson City, TN Body: Cytomegalovirus (CMV) infection in the neonate is an infrequent occurrence in the developing world, and observing the symptoms of ocular CMV infection such as vitritis is rare. Treating CMV infection promptly is necessary to prevent mortality and potential neurological deficits including blindness and hearing loss. We encountered a preterm infant presenting with CMV sepsis immediately after birth. Our question was: will the current standard of treatment for CMV sepsis prevent CMV ocular infection? With our method of treatment, we followed the current standard of treatment for CMV infection by administering intravenous Gancyclovir for 6 weeks and oral Valgancyclovir for 6 months. Despite using the standard treatment to prevent neurological sequelae, the patient developed CMV vitritis and retinitis bilaterally. Although the treatment did not prevent CMV ocular infection, the severity of CMV retinitis and vitritis improved with treatment, and full resolution of vitritis was noted by day of life 61.

INTRODUCCIÓN: La patología de la superficie ocular puede requerir de biosustitución mediante el implante de diferentes tipos de tejido. Se han descrito diversos biosustitutos de la superficie ocular. Las indicaciones, técnicas quirúrgicas, formulaciones y controles de calidad han sufrido modificaciones y cambios en los últimos años. HIPÓTESIS: Existe un cambio de tendencias en las indicaciones y formulaciones y/o técnicas quirúrgicas para el manejo y tratamiento de la patología de la córnea y superficie ocular. Asimismo, se considera que el control de la calidad de estos biosustitutos es de suma importancia para evitar efectos adversos y complicaciones para el receptor. Además, la investigación y desarrollo constantes de los biosustitutos de origen humano, ha permitido recientemente disponer de nuevos formatos y formulaciones para la aplicación clínica en el tratamiento de diferentes patologías de la superficie ocular OBJETIVO: Conocer las indicaciones de biosustitutos de origen humano - córnea, esclera, membrana amniótica - para el tratamiento de las patologías de la córnea y superficie ocular en Catalunya. Evaluar el uso de los cultivos microbiológicos de anillos esclerocorneales y medios de conservación corneal como control perioperatorio del tejido donante en el mundo real. Evaluar los resultados clínicos de los biosustitutos autólogos, como la conjuntiva, y evaluar los resultados clínicos de los recientes formatos de la membrana amniótica disponibles en Cataluña – liofilizada y extracto de membrana amniótica – desde un punto de vista de validación y estudio en la práctica clínica. RESULTADOS: Las indicaciones más frecuentes en Catalunya para queratoplastia entre los años 2011-18 son queratopatía bullosa, distrofia endotelial de Fuchs y retrasplante. La queratoplastia penetrante es aún la técnica más prevalente a pesar de que se ha encontrado una tendencia creciente estadísticamente significativa de las queratoplastias endoteliales y especialmente para la Descemet Membrane Endothelial Keratoplasty, en estrecha relación con la disponibilidad de tejido precortado. Las principales indicaciones del implante de tejido escleral en Catalunya entre los años 2011-18 son la cirugía del glaucoma y la reconstrucción palpebral. Y para la utilización de la membrana amniótica en Catalunya entre los años 2011-18 son las úlceras corneales y la reconstrucción conjuntival. Con relación a los controles microbiológicos de las córneas utilizadas para queratoplastia, las técnicas de preservación de tejido mediante las aproximaciones en hipotermia muestran una mayor tasa de contaminación de los tejidos, estadísticamente significativa, que las aproximaciones en cultivos organotípicos. El estudio de la adherencia al tratamiento postoperatorio tras cirugía de pterigión con autoinjerto conjuntival muestra que la adherencia está relacionada de manera estadísticamente significativa con el protocolo de tratamiento. La membrana amniótica es un biosustituto que ha demostrado tener efectos beneficiosos tanto en la presentación liofilizada - como coadyuvante para la reconstrucción de la superficie ocular en la cirugía del pterigión -, como en forma de colirio de extracto - para el cierre epitelial y en la disminución de la sintomatología asociada al ojo seco tanto es un entorno controlado como en un estudio de vida real. Conclusión: Las técnicas de queratoplastia lamelares aún no son las predominantes en nuestro medio, a pesar de que ha habido un cambio de tendencias importante y en relación con la disponibilidad de tejido precortado. Las principales indicaciones del implante de tejido escleral son la cirugía del glaucoma y la reconstrucción palpebral; y las principales indicaciones de la utilización de la membrana amniótica son las úlceras corneales y la reconstrucción conjuntival. Las técnicas de preservación de tejido corneal mediante las aproximaciones en hipotermia muestran una mayor tasa de contaminación. Los pacientes tras cirugía de pterigión con autoinjerto conjuntival presentan diferente tasa de adherencia al tratamiento postoperatorio según el protocolo postoperatorio indicado. La membrana amniótica es un biosustituto que ha demostrado tener efectos beneficiosos tanto en la presentación liofilizada - como coadyuvante para la reconstrucción de la superficie ocular en la cirugía del pterigión -, como en forma de colirio de extracto - para el cierre epitelial y en la disminución de la sintomatología asociada al ojo seco.
Ocular surface patologies treatment could require of substitutes of human origin (SoHO). Diferent SoHO have been described and its indications, surgical techniques, formulations and quality controls have been modified for the last years. The objectives of this investigation were to know the indications in Catalonia for SoHO for ocular surface treatment, to evaluate the microbiological cultures as a perioperative quality control and evaluate clinical outcomes of autologous and allogenic SoHO in Catalonia. RESULTS: main indications of queratoplasty from 2011 to 2018 were bullous keratoplasty, Fuchs endothelial dystrophy and regraft. Penetrant keratoplasty was the principal technique despite a statistical significant ascendent trend in lamellar keratoplasties, related to precut tissue availability. Main indications of sclera tissue were glaucoma surgery and lid reconstruction and of amniotic membrane were corneal ulcer and conjunctival recontruction. Corneas preserved in hypothermia had a statistical significant higher contamination rate. Adherence to postoperative treatment in patients with previous pterigium surgery is statistically related to the protocol. Amniotic membrane showed to have beneficial effects in its different formulations – liophylized and as a amniotic membrane extract eye drops for different patologies as wound healing defects, dry eye disease and pterigium. CONCLUSION: lamelar keratoplasty techniques have not yet achived the predominance in our area despite a statistical significant shift in trends has been detected indeed. Main indications of keratoplasty were bullous keratoplasty and Fuchs endothelial dystrophy; of scleral tissue were glaucoma surgery and lid reconstruction and of amniotic membrane were corneal ulcer and conjunctival recontruction. Corneas preserved in hypothermia showed a higher contamination rate. Adherence to postoperative treatment in patients with previous pterigium surgery is statistically related to the protocol. Amniotic membrane showed to have beneficial effects in its different formulations – liophylized and as a amniotic membrane extract eye drops.

El eje cerebro-intestino es una comunicación bidireccional entre ambos órganos que incluye mediadores inmunológicos, nerviosos, endocrinos y respuesta microbiana. Una desregulación de esta comunicación podría estar detrás de enfermedades como la enfermedad de Párkinson, la enfermedad de Alzheimer, la depresión, o incluso el ictus o infarto cerebral. El ictus se produce por la interrupción brusca de la circulación sanguínea en una zona de cerebro, y es la primera causa de muerte en mujeres y primera causa de discapacidad adquirida en adultos. El campo de investigación de la relación cerebro- intestino en el contexto del ictus es reciente, con un aumento de las publicaciones en los últimos cinco años. En estas investigaciones se ha constatado que el ictus es capaz de modificar la composición bacteriana intestinal, que la alteración de la microbiota intestinal mediante intervención externa (como uso de tratamientos antibióticos o trasplantes fecales) puede modular la lesión cerebral vía respuesta inmune y que el ictus actúa sobre la integridad de la barrera intestinal. Dada la corta vida de este campo, quedan varios aspectos que abordar, resolver y aclarar, ya que entre las diferentes investigaciones hay resultados controvertidos y aspectos de la respuesta inmunológica en el intestino que no se conocen. Con el objetivo de esclarecer los efectos del ictus sobre el intestino, en esta tesis doctoral se han utilizado modelos experimentales de isquemia cerebral en ratón y se han analizado los cambios de las poblaciones celulares del sistema inmune después del ictus mediante citometría de flujo. Se ha estudiado el efecto de la isquemia cerebral sobre las poblaciones bacterianas intestinales mediante PCR cuantitativa y sus metabolitos por cromatografía de gases, y la regulación de las mismas mediante tratamientos para el bloqueo de la vía adrenérgica. Además, se ha estudiado la integridad física de la barrera intestinal mediante técnicas de análisis de permeabilidad in vivo y ex vivo. Los resultados muestran que después del ictus se produce un aumento a nivel de linfocitos intraepiteliales de células Tγδ, implicadas en el mantenimiento de la integridad de la barrera intestinal, además de un incremento de la citocina proinflamatoria IFNγ en ganglios mesentéricos, situación que se evita previo tratamiento con el β-bloqueante propranolol. De forma consistente, observamos un aumento de la familia de enterobacterias en muestras de heces y de ciego de los animales que habían sufrido un ictus, además de un aumento de la detección del número de colonias bacterianas en muestras de ganglios mesentéricos, situación no dependiente de la vía adrenérgica. En paralelo a estos resultados de translocación bacteriana, confirmamos presencia de bacterias bioluminiscentes procedentes del tracto intestinal en pulmones de animales isquémicos. Finalmente, también hemos comprobado que la isquemia aumenta la secreción iónica intestinal en tiempos cortos después del ictus, y que además podría producir pequeñas alteraciones de las uniones celulares del epitelio intestinal.
The gut-brain axis is a bidirectional communication between both organs that includes immune, nervous, endocrine mediators and a microbial response. Disruptions in this communication could trigger diseases such as Parkinson's disease, Alzheimer's disease, depression, or even stroke, which is caused by the interruption of the brain blood circulation. The research between the gut and the brain in the context of stroke is recent, with most of the research performed in the last 4 years. As a consequence, there are several aspects to be addressed, resolved and clarified. In order to clarify the effects of stroke on the intestine, we performed the stroke experimental model of transient middle cerebral artery occlusion in mice and analysed changes in the intestinal immune system populations by flow cytometry. We also studied the effect of the cerebral ischemia on the gut bacteria and its metabolites by qPCR and gas chromatography, respectively, as well as its regulation by drug treatments. Furthermore, we identified changes in gut epithelial integrity trough in vivo and ex vivo permeability tests. Our results show an increase in intraepithelial γδ T cells, involved in maintaining the integrity of the intestinal barrier, in addition to an increase in the proinflammatory cytokine IFNγ in mesenteric lymph nodes, what ultimately is avoided after treatment with the β-blocker propranolol. Consistently, we observed an increase in the Enterobacteriaceae family in stool and cecum samples from stroke animals. In addition, we observed higher numbers of bacterial colonies in samples of mesenteric lymph nodes from stroke animal in comparison to control animals, a situation that was not dependent on the adrenergic pathway. This results suggested some bacterial translocation and, accordingly, we confirmed the presence of bioluminescent bacteria from the intestinal tract in the lungs of ischemic animals. Finally, we also detected alterations in the intestinal epithelium after stroke, such as an increase in intestinal ion secretion, and changes in the gut permeability, suggesting that ischemia could produce small alterations in the cellular junctions of the intestinal epithelium. These results show the relationship between the brain and the intestine after stroke and the possibility to regulate this response by pharmacological treatment.

P2X receptors and pannexins (Panx) are eukaryotic ion channels that are implicated in a range of diseases and conditions including cancer, inflammation and pain sensation and as a result, are important therapeutic targets. Deducing their 3D-structures would enable the use of structure-based drug design to identify novel agonists or antagonists. However, solving eukaryotic membrane protein structures is a significant challenge due to the requirement for high yields of purified folded, functional protein, which are not readily obtainable with conventional over-expression systems. By using P2X receptors and pannexins as model ion channel targets, this thesis aims to test Drosophila melanogaster as a system for the over-expression and functional analysis of eukaryotic ion channels. A number of epitope-tagged P2X and Panx protein constructs were generated and first expressed in HEK-293 cells (rat P2X2-GFP, human P2X4-GFP, rat Panx1-GFP and human P2X4-int-CBD (chitin binding domain)) to allow their expression, glycosylation and oligomeric states to be investigated as markers of protein folding and quality. Subsequently, rat P2X2-GFP and rat Panx1-GFP constructs were successfully expressed in the photoreceptor cells of Drosophila melanogaster, where the photoreceptive membrane in the visual system is organised into a densely packed brush of microvilli, the rhabdomere. This system provides a large surface area of membrane for protein expression. Although the yields of purified protein were lower than expected, rat Panx1-GFP was successfully purified and used for low resolution structural studies with transmission electron microscopy. Rat P2X2-GFP was also expressed in the nervous system of Drosophila under control of a pan-neural, C155-Gal4 driver and was shown to be functional by measuring ATP-evoked action potentials using electrophysiological recordings of the Drosophila taste sensilla. This system was also used to test the activity of an adenosine nucleotide library of 80 compounds. Three nucleotides were identified that elicited responses similar to ATP; these were 2F-ATP, ATPαS and ATPγS.

Escherichia coli is a natural inhabitant of the intestines of both humans and animals, but there are also several pathogenic types of E. coli which cause disease in humans.

Strains of enterohemorrhagic E. coli (EHEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of shigatoxin 1 and 2 or combination of these toxins. Other major virulence factors include EHEC hemolysin and intimin, the product of the eae gene that is involved in attaching and effacing adherence phenotype. EHEC has also been associated with uncomplicated diarrhea.

The capacity to control EHEC disease and to limit the scale of outbreaks is dependent upon prompt diagnosis and identification of the source of infection.

The principal reservoirs of EHEC are cattle and food products, which presumably have come into contact with domestic animal manure and/or are inadequately pasteurised, these are important vehicles of infection.

In the present study, the PCR technique with primers detecting the verocytotoxin genes was shown to be a possible method to screen for and identify EHEC.

In summary stx genes were detected in 16 samples of 228 sampels and the eae gene was detected in 2 samples using PCR.

Orientador: Meuris Gurgel Carlos da Silva
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica
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Resumo: O lodo de esgoto, um dos principais componentes das águas residuárias geradas mundialmente, é um resíduo com alto conteúdo de umidade e grande carga de microrganismos. O uso da secagem apresenta-se como uma poderosa ferramenta na área ambiental devido a possibilidade de redução de volume e inativação microbiológica. O presente trabalho teve como objetivo avaliar o processo de secagem empregando um secador convectivo direto de fluxo ascendente, para redução de volume e inativação microbiológica de lodo de ETE. O lodo utilizado foi oriundo do tratamento primário da ETE - Tatu, da cidade de Limeira - SP. Foram desenvolvidos os seguintes estudos: determinação das características físico-químicas, ensaios de secagem a partir do planejamento experimental 3² em duplicata (duas variáveis - temperatura e vazão do ar de secagem, e três níveis) com análise estatística dos dados, a avaliação da redução de volume do lodo seco, ajuste de modelos matemáticos aos dados experimentais, verificação do efeito da sazonalidade do lodo de esgoto no processo de secagem, e avaliação da inativação microbiológica. Pelos resultados do trabalho verificou-se que as características físicoquímicas não sofreram alterações significativas antes e após o processo de secagem, e com isso, pode-se considerar que não ocorreu emissão atmosférica destes componentes. A cinética do processo de secagem se caracterizou inicialmente pelo período de aquecimento do material seguindo-se pelos períodos de secagem à taxa constante, 1.ª e 2.ª taxas decrescentes. O início do 2.º período de taxa decrescente foi marcado pela quebra ou fissura da torta, com aumento significativo da taxa de secagem, que é uma característica específica da secagem de materiais como o lodo. A análise estatística mostrou que a temperatura foi a variável mais significativa, indicando que o mecanismo de secagem do lodo de esgoto foi predominantemente difusivo. Contudo, devido o comportamento do 2.º período de secagem, os modelos difusivos não se ajustaram adequadamente, necessitando de modelos empíricos para descrever a 2.ª taxa decrescente. A redução de volume do material foi satisfatória, no caso cerca de 50%. A verificação da sazonalidade mostrou uma discreta diferença nos valores de pH e umidade inicial dos lodos estudados. As análises microbiológicas mostraram que após o tratamento térmico, houve a inativação microbiológica em praticamente todas as condições de processo estudadas, sendo o binômio tempo de exposição - temperatura o fator predominante para a esterilização e desinfecção do lodo de ETE. Com os resultados obtidos é possível considerar que o processo de secagem deste trabalho apresenta bom potencial de aplicação como tratamento de lodo de ETE, mas também de outros materiais que possuam características similares. Palavras-chave: secagem, lodo de esgoto, redução de volume, inativação microbiológica.
Abstract: Sludge, one of the main components of wastewater generated worldwide, is a waste with great quantity of moisture and microorganisms. The usage of drying has been a powerful tool in the environmental area due to the possibility of reduction of volume and microbiological inactivation. The present study aims to assess the drying process using an upflow direct convection dryer to reduce sludge volume and microbiological inactivation of Sludge Treatment Plants (STP). The used sludge has been taken from the primary treatment of STP - Tatu, city of Limeira, São Paulo. The following studies have been carried out: determination of physicochemical characteristics, drying experiments from the experimental design 3² in duplicate (two variables - temperature and drying air flow, and three levels) with statistical analysis of data, assessment of volume reduction of dried sludge, fitting of mathematical models to experimental data, verification of the effect of the seasonality of the sludge in the drying process, and assessment of microbiological inactivation. With the results of this study it could be verified that the physicochemical characteristics have not suffered significant changes nor before neither after the drying process, therefore, it can be said that atmospheric emission of these components have not occurred. The kinetics of the drying process was initially characterized by the heating period of the material followed by the drying periods of constant rate, first and second falling rates. The beginning of the second period of falling rate was marked by the cake break or fissure with the significant increase of the drying rate which is a specific characteristic of the drying of materials such as sludge. The statistic analysis has shown that the temperature was the most significant variable indicating that the sludge drying mechanism has predominantly been diffusive. However, due to the behavior of the second period of drying, the diffusive models did not adjust adequately, being necessary the use of empiric models to describe the second falling rate. The reduction of material volume was satisfactory, 50%, in this case. The verification of the seasonality has shown a discrete difference in the pH values and in the moisture content of the studied sludge. The microbiological analyses showed that after the thermal treatment there was microbiological inactivation practically in all the studied conditions of the process, being the binomial time of exposition - temperature the predominant factor for the sterilization and disinfection of sludge of STP. With the obtained results it is possible to consider that the drying process of this study shows a good potential of application for sludge treatment of STP, and also for other materials that have similar characteristics.
Doutorado
Engenharia de Processos
Doutor em Engenharia Química

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Decontamination of the surface of the donor eyeballs is part of the operational norms that eye banks advocate before preservation, and antisepsis procedures are effective, ensuring greater transplantation safety. The objective of the present study was to evaluate the antiseptic effect in reducing the microbiota of the ocular globe of donors of corneas prior to enucleation, with 5% povidone-iodine (PVP-I) and 0.05% chlorhexidine gluconate (GC), In the action times of 5, 10 and 15 minutes, as well as the susceptibility profile of the microbiota isolated from gentamicin. Thirty pairs of corneas received antiseptics, with PVP-I in the right eye and GC in the left, and for each time of action 10 pairs of eyeballs were used. Swabs were collected from the ocular surface before application of the solutions, after and at the time of preservation of the corneal tissue, to evaluate the reduction of the microbiota. After identification of the microbiota, an antibiogram test was performed with gentamicin. The data were computed and evaluated by Chi-square or Fisher's exact test, T-test and McNemar test paired, and the statistical significance level was 5% (p <0.05). In the second collection, after antisepsis, there was a reduction of 39,5% in the total of gram positive bacteria (G +), and of 76,5% in the gram negative (G-) bacteria, with no statistically significant difference (p = 0.183), which demonstrated that the bacterial elimination capacity of the antiseptics was similar. It was observed that, in the second collection, both were more effective for G-, with a statistically significant difference (p <0.001), than for G +, with no statistically significant difference (p = 0.494). In the third collection, after the residual effect of the antiseptics, there was a reduction of 99.1% of all the microorganisms. In the antibiogram test, 88% of the isolated microorganisms were sensitive to gentamicin. It was concluded that the use of antiseptics is essential for the effective decontamination of donated corneas prior to preservation. The residual time of the antiseptics increased the decontamination power of PVP-I and GC, being similar in reducing the microbiota of the ocular globe of the donor of corneas. Gentamycin contained in the cornea preservation medium complements the antisepsis of the donated tissues.
A descontaminação da superfície dos globos oculares doados são normas operacionais que os bancos de olhos preconizam antes da preservação e os procedimentos de antissepsia são eficazes, garantindo uma maior segurança ao transplante. O objetivo do presente estudo foi avaliar o efeito antisséptico na redução da microbiota do globo ocular de doadores de córneas antes da enucleação, com o povidona-iodo (PVP-I) a 5% e gluconato de clorexidina (GC) a 0,05%, nos tempos de ação de 5, 10 e 15 minutos, bem como o perfil de susceptibilidade da microbiota isolada à gentamicina. Trinta pares de córneas receberam antissépticos, sendo o PVP-I no olho direito e o GC no esquerdo, e para cada tempo de ação foram utilizados 10 pares de globos oculares. Foram colhidos swabs da superfície ocular antes da aplicação das soluções, após e no momento da preservação do tecido corneano, para avaliar a redução da microbiota. Após identificação da microbiota, foi realizado teste de antibiograma com gentamicina. Os dados foram computados e avaliados pelos testes Qui-Quadrado ou Exato de Fisher, teste T e Teste McNemar pareado, e o nível de significância estatística foi (p<0,05). Com relação aos dados obtidos na segunda coleta, após o uso de antissépticos, houve uma redução de 39,5% no total de bactérias gram positivas (G+) e de 76,5% nas gram negativas (G-), não havendo diferença estatística significativa (p=0,183), sendo semelhante a capacidade de eliminação bacteriana dos antissépticos. Observa-se que, na segunda coleta, ambos foram mais eficazes para as G-, com diferença estatisticamente significativa (p<0,001), do que para as G+, sem diferença estatisticamente significativa (p=0,494). Na terceira coleta, após o efeito residual dos antissépticos, houve redução de 99,1% de todos os micro-organismos. No teste de antibiograma, 88% dos micro-organismos isolados foram sensíveis à gentamicina. Concluiu-se que o uso de antissépticos é essencial para a efetiva descontaminação das córneas doadas antes da preservação. O tempo residual dos antissépticos aumentou o poder de descontaminação do PVP-I e GC, sendo semelhantes na redução da microbiota do globo ocular do doador de córneas. A gentamicina contida no meio de preservação de córnea complementa a antissepsia dos tecidos doados.

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O presente estudo foi realizado no período de janeiro de 2012 a janeiro de 2013 em fazendas leiteiras da região de Dracena, São Paulo. Durante o período, foram coletadas 800 amostras de fezes com suabes retais em vacas leiteiras. Essas amostras foram levadas para o Laboratório de Microbiologia do Campus Experimental de Dracena, onde foram isoladas e identificadas 561 amostras para Escherichia coli. Após o isolamento foram extraídos os DNAs de todas as amostras pelo método da fervura e por PCR o DNA foi amplificado para se detectar a presença dos genes de virulência de E. coli pertencentes ao grupo STEC, produtora de toxina tipo shiga em 446 amostras. De todas as cepas isoladas 90 eram portadoras do gene stx1, 97 do gene stx2, 45 do gene eae, 37 dos genes stx1 e stx2, 110 dos genes stx1 e eae e 67 dos genes stx2 e eae. Foram isoladas também 115 cepas que não eram portadoras de nenhum dos genes de virulência de STECs do estudo. Todos os isolados de E. coli portadores de cada gene de virulência foram avaliados quanto a resistência frente a 10 antimicrobianos. Os percentuais de resistências aos antimicrobianos foram maiores para a lincomicina, penicilina e novobiocina e menores para ampicilina, neomicina e tetraciclina. Foram identificados os sorogrupos, dos quais os mais frequentes entre os isolados portadores do gene de virulência stx1 foram o O119 e O114; do gene de virulência stx2 foram os sorogrupos O9 e O8; e do gene de virulência eae foram os sorogrupos O9, O8 e O127. Todos os isolados de E. coli apresentaram multirresistência e a maioria apresentou maior percentagem de multirresistência contra 2 a 3 e contra 10 antimicrobianos. Não foi verificado estatisticamente relação entre os padrões de virulência e os padrões de resistência aos antimicrobianos entre as amostras
The present study was conducted between january 2012 to january 2013 on dairy farms of Dracena city region, São Paulo. During the period, 800 samples of faeces were collected with rectal suabs from dairy cattle cows. Those samples were taken to the laboratory of microbiology of Dracena Experimental Campus, where 561 samples were isolated and identified for Escherichia coli. After the DNA from the samples were extracted by the boiling method and with PCR the genetic material was amplified to detect the presence of virulence genes from STEC, shiga-like toxin producer E. coli, on 446 samples. Of those samples, 90 were carriers of the stx1 gene, 97 of the gene stx2, 45 of the gene eae, 37 of the genes stx1 and stx2, 110 of the genes stx1 and eae, 67 of the genes stx2 and eae. Also were isolated 115 samples that did not carry none of the virulence genes from STECs of the study. All the E. coli isolates of each virulence gene were evaluated for resistence to 10 antibiotics. The percentual of resistence were higher for lincomycin, penicillin and novobiocin and lower for ampicillin, neomycin and tetracycline. A serogroup test was made, of which the most frequent among isolates carrying the virulence gene stx1 were O119 and O114; of the gene stx2 were serogroups O8 and O9; and of the gene eae were the serogroups O9, O8 and O127. All the E. coli isolates presented multirresistence and most isolates presented more percentage of multirresistence against 2 to 3 and against 10 antibiotics. Was not verified statistically relationship between virulence patterns and patterns of antimicrobial resistance among the samples

Orientador: Everlon Cid Rigobelo
Banca: Hélio José Montassier
Banca: José Carlos Rende
Resumo: O presente estudo foi realizado no período de janeiro de 2012 a janeiro de 2013 em fazendas leiteiras da região de Dracena, São Paulo. Durante o período, foram coletadas 800 amostras de fezes com suabes retais em vacas leiteiras. Essas amostras foram levadas para o Laboratório de Microbiologia do Campus Experimental de Dracena, onde foram isoladas e identificadas 561 amostras para Escherichia coli. Após o isolamento foram extraídos os DNAs de todas as amostras pelo método da fervura e por PCR o DNA foi amplificado para se detectar a presença dos genes de virulência de E. coli pertencentes ao grupo STEC, produtora de toxina tipo shiga em 446 amostras. De todas as cepas isoladas 90 eram portadoras do gene stx1, 97 do gene stx2, 45 do gene eae, 37 dos genes stx1 e stx2, 110 dos genes stx1 e eae e 67 dos genes stx2 e eae. Foram isoladas também 115 cepas que não eram portadoras de nenhum dos genes de virulência de STECs do estudo. Todos os isolados de E. coli portadores de cada gene de virulência foram avaliados quanto a resistência frente a 10 antimicrobianos. Os percentuais de resistências aos antimicrobianos foram maiores para a lincomicina, penicilina e novobiocina e menores para ampicilina, neomicina e tetraciclina. Foram identificados os sorogrupos, dos quais os mais frequentes entre os isolados portadores do gene de virulência stx1 foram o O119 e O114; do gene de virulência stx2 foram os sorogrupos O9 e O8; e do gene de virulência eae foram os sorogrupos O9, O8 e O127. Todos os isolados de E. coli apresentaram multirresistência e a maioria apresentou maior percentagem de multirresistência contra 2 a 3 e contra 10 antimicrobianos. Não foi verificado estatisticamente relação entre os padrões de virulência e os padrões de resistência aos antimicrobianos entre as amostras
Abstract: The present study was conducted between january 2012 to january 2013 on dairy farms of Dracena city region, São Paulo. During the period, 800 samples of faeces were collected with rectal suabs from dairy cattle cows. Those samples were taken to the laboratory of microbiology of Dracena Experimental Campus, where 561 samples were isolated and identified for Escherichia coli. After the DNA from the samples were extracted by the boiling method and with PCR the genetic material was amplified to detect the presence of virulence genes from STEC, shiga-like toxin producer E. coli, on 446 samples. Of those samples, 90 were carriers of the stx1 gene, 97 of the gene stx2, 45 of the gene eae, 37 of the genes stx1 and stx2, 110 of the genes stx1 and eae, 67 of the genes stx2 and eae. Also were isolated 115 samples that did not carry none of the virulence genes from STECs of the study. All the E. coli isolates of each virulence gene were evaluated for resistence to 10 antibiotics. The percentual of resistence were higher for lincomycin, penicillin and novobiocin and lower for ampicillin, neomycin and tetracycline. A serogroup test was made, of which the most frequent among isolates carrying the virulence gene stx1 were O119 and O114; of the gene stx2 were serogroups O8 and O9; and of the gene eae were the serogroups O9, O8 and O127. All the E. coli isolates presented multirresistence and most isolates presented more percentage of multirresistence against 2 to 3 and against 10 antibiotics. Was not verified statistically relationship between virulence patterns and patterns of antimicrobial resistance among the samples
Mestre

Chez la guêpe parasitoïde Venturia canescens, des particules virales dépourvues d'ADN appelées VLP (pour "Virus-like Particules") sont produites spécifiquement dans les ovaires et tapissent le chorion des oeufs qui sont injectés dans la chenille hôte. Les VLP ont une fonction immunosuppressive pour l'hôte parasité et permettent ainsi la survie des oeufs du parasitoïde. Ces VLP résultent de l’intégration d’un nudivirus dans le génome de l’ancêtre de la guêpe, nudivirus qui a été ensuite domestiqué pour former des liposomes viraux capables de véhiculer dans l’hôte des protéines de virulence d'origine cellulaire. L’étude réalisée au cours de cette thèse a eu pour objet, d’une part, d'étudier les mécanismes de domestication virale qui ont conduit au virus symbiotique endogène actuel nommé VcENV (pour V. canescens endogenous nudivirus) et d’autre part, d'apporter des éléments de réponse sur le processus de morphogénèse et le mode d'action parasitaire des VLP
Viral particles devoid of DNA called VLPs (for Virus-Like Particles) are specifically produced in the ovaries of the parasitoid wasp Venturia canescens and line the chorion of the wasp’s eggs injected into the host caterpillar. VLPs are immunosuppressive and allow parasitoid eggs survival. These VLPs result from the integration of a nudivirus into the wasp ancestor genome, nudivirus which was then domesticated to form viral liposomes capable of carrying, into the host, virulence proteins of cellular origin. The aim of the study carried out during this thesis was, first, to analyze the viral domestication mechanisms that led to the current endogenous symbiotic virus called VcENV (for V. canescens endogenous nudivirus) and secondly to provide some answers on VLPs morphogenesis process and parasitic mode of action

Corneal endothelial development is an intricate process driven by finely tuned gene expression. Its formation is necessary for the continued normal development of the anterior segment of the eye. The presence of an inductive lens able to secrete factors such as TGFβ2 as well as the expression of Foxc1 and Pitx2 is essential to corneal endothelial development, as in the absence of any of these; the corneal endothelium fails to form. Corneal endothelial development begins as peri-ocular mesenchyme (POM) cells migrate into the space between the lens and surface ectoderm at E11.5. From E12.5, these cells begin to transition from a mesenchymal to an epithelial/endothelial (MET) phenotype, differentiating into a monolayered endothelium by E15 characterised by inter-cellular junctions. To study the initial process of development, immortalised POM cell lines from E12.5 and E13.5 embryos were used. Expression of the key genes, the transcription factors, Foxc1 and Pitx2 and two genes involved in EMT/MET, Slug and Tsc22, were analysed at these stages to establish the developmental norm. The effect of the lens on these expression levels was then determined. To establish whether TGFβ2 is the lens secreted signal responsible for gene expression changes, cells were subjected to TGFβ2 treatment. In all these experiments, the role of Foxc1 in regulating gene expression was determined by Foxc1 overexpression and knockdown. The effect of the lens on cellular proliferation and on the expression and cellular arrangement of N-cadherin, a junction protein was also determined. The results showed that, at E12.5, the lens downregulates Foxc1 and Pitx2 expression, is a potent inducer of Tsc22 expression and is required for maintaining Slug levels. TGFβ2 was shown to play a role in Foxc1 and Pitx2 downregulation. Analysis suggests that Tsc22 expression is responsive to lens signals, but that TGFβ2 is not the signal responsible for its downregulation between E12.5 and E13.5. The lens has no effect on Slug expression in the presence of Foxc1, but when Foxc1 is silenced, Slug is induced. Thus, Foxc1 plays a crucial regulatory role in Slug expression. At E13.5, as differentiation is initiated, Foxc1 expression remains responsive to the lens and to TGFβ2. Pitx2 expression is still induced by the lens but, at this stage, TGFβ2 does not play a part in Pitx2 regulation suggesting involvement of other unknown lens secreted signals. Other lens secreted signal/s were also shown to downregulate Tsc22 and Slug at this stage. The lens was implicated in MET as it was shown to have an effect on N-cadherin localisation in 3-dimensional culture. E12.5 Spheroids exposed to E6 lenses formed a distinct lattice arrangement of N-cadherin compared to the uniform distribution in control cells. Although the 13.5 control cell aggregates also showed a lattice framework, it was more pronounced in the lens treated cells. The transcriptional role of Foxc1 was determined by overexpression and knockdown experiments where Foxc1 overexpression and knockdown upregulated Tsc22 and downregulated Pitx2 and Slug at E12.5. At E13.5, Pitx2 was downregulated and Slug was upregulated in response to aberrant expression of Foxc1. This was illustrative of the sensitivity these genes have to Foxc1 expression during development. It is known that the presence of a functioning lens and Foxc1 are essential for proper development of the corneal endothelium, which in turn is necessary for normal eye development. The understanding of the precise molecular mechanisms required for corneal endothelial development and the processes requisite for cell proliferation and differentiation has important consequences for providing further insight into the pathophysiology of anterior segment dysgenesis and glaucoma. Previous studies suggest that stem-cell like qualities are conferred in cells undergoing EMT. Such an investigation may lead to application in regenerative medicine such as the bioengineering of corneal tissue.
Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

The proper organization and differentiation of the anterior segment is pivotal for normal eye development. Neural crest-derived POM cells are key contributors to correct anterior segment formation, differentiating to form the monolayered corneal endothelium. Mice with homozygous null mutations in the forkhead transcription factor gene, Foxc1, fail to develop a proper corneal endothelium stabilized by adherens junctions, with the endothelium adhering to the lens, preventing anterior chamber separation. The aim of this study was to evaluate the interaction between Foxc1 and the adherens junction protein, N-cadherin, as well as an associated gene, Msx1, during key stages in corneal endothelium development. Foxc1 was over-expressed in E12.5 and E13.5 POM cells and qPCR was carried out to determine the effect of Foxc1 on N-cadherin and Msx1 gene expression. Data showed over-expression of Foxc1 in wildtype E12.5 and E13.5 POM cells to cause significant fluctuations in N-cadherin and Msx1 expression (p < 0.05). POM cells were then transfected with a Foxc1 knock-down plasmid or the Foxc1 overexpression plasmid to evaluate the effect of Foxc1 on N-cadherin protein expression by Western blot analysis, however, these results were inconsistent with the gene expression analyses with no significant differences in N-cadherin expression detected. N-cadherin protein expression and localization was then further assessed by means of immunocytochemistry (ICC) and confocal microscopy in monolayer and hanging-drop POM cell cultures. Both qPCR and confocal microscopy data showed consistency, indicating increased amounts of N-cadherin in E12.5 cells relative to E13.5 cells, with membrane-bound N-cadherin showing a clear lattice-work pattern in hanging drop culture. Foxc1 over-expression/knock-down studies on E12.5 and E13.5 POM cells together suggest that N-cadherin is transcriptionally regulated by Foxc1 and that Foxc1 has a threshold level at which it is able to exert control over N-cadherin in POM cells. Foxc1 expression is therefore essential in establishing N-cadherin adhesion junctions in the corneal endothelium. Preliminary data also suggests that Msx1 may directly interact with Foxc1 in POM cells, however, further studies must be undertaken to verify and establish the effects of Foxc1/N-cadherin/ Msx1 interaction in the development of a cohesive, integrated corneal endothelium and functional anterior segment.
Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Relatório de Estágio do Mestrado em Biologia Clínica Laboratorial
As análises laboratoriais têm um grande impacto nas decisões tomadas pelas equipas médicas dado que fornecem dados essenciais para um correto diagnóstico e atribuição de terapêutica. O presente relatório pretende descrever todas as atividades realizadas durante 8 meses de estágio no Serviço de Patologia Clínica do “Hospital Santa Maria Maior” nas áreas da Microbiologia, Hematologia/Coagulação, Imunoquímica (composta pela Bioquímica, Imunologia e Virologia) e Imuno-Hematologia (banco de sangue). Nas diversas áreas referidas pretende-se descrever todas as fases que compõem o estudo laboratorial, mais concretamente as metodologias, equipamentos e materiais utilizados na rotina laboratorial diária para o estudo de parâmetros/produtos/analitos, para assim chegar a uma ou mais patologias. Tendo sempre como referência o trabalho do hospital desde o acompanhamento de pacientes do internamento, do serviço de urgência, ao hospital de dia, consultas externas e centros de saúde da região de Barcelos.
Laboratory analyses have a great impact on the decisions made by medical teams, since they provide the essential data to a correct diagnosis and choice of therapy. This report aims to describe all the activities carried out during the 8 months of internship in “Santa Maria Maior Hospital” Clinical Pathology Service in the fields of Microbiology, Hematology/Coagulation, Immunochemistry (composed of Biochemistry, Immunology and Virology) and Immunohematology (blood bank). In the above referred areas, this report intends to describe all the phases that comprise the laboratory study, namely the methodologies, equipment and materials used, during the daily laboratory routine for the study of all the parameters / products / analytes, in order to arrive at one or more pathologies. These approaches were always referred the hospital base work, i.e. the follow-up of patients, the emergency service, day hospital and health centers in the Barcelos region.

A series of grazing experiments were conducted in the summer/autumn of 2003 and 2004 at Massey University's Riverside dryland farm near Masterton in Wairarapa on the East Coast of NZ, to study the effects of grazing willow fodder blocks (6,000 stems/ha) upon the production and reproductive performance of ewes relative to ewes grazing drought pastures. Drought pastures were simulated in this study and included short drought pasture and long drought pasture. Pasture with a low pre-grazing mass of approximately 1500 kg OM/ha, a dead matter content of >50 % and a sward height of 5-7 cm was defined as short drought pasture typical of drought conditions. Long drought pasture was similar to pasture growing in the willow fodder blocks, with a pre-grazing pasture mass of >4000 kg OM/ha, a sward height of > 30cm and a dead matter content of 30-60 % . Willow fodder blocks were established on low-lying wet, marshy areas of the farm that had very low or zero productivity in the undeveloped state. Pasture development in the fodder blocks was noticed with the growth of unsown grasses and legumes, as the areas dried up following the planting of willow stakes, due to evapotranspiration from the trees. Forage in the willow fodder blocks included both trees and pasture that was grown under the trees. The nutritive value of short drought pasture was low with an ME of 8 MJ/kg O M ; long drought pasture ranged between 8- 1 0 MJ ME/kg DM; willow pasture contained 8 MJ M Elkg DM in 2003 and 1 0 MJ ME/kg OM in 2004. The nutritive value of edible willow tree (<5 mm diameter) was superior to drought pasture with an ME of > 10 MJ/kg OM. The concentrations of the secondary compounds such as condensed tannins (CT ; 30- 40 glkg OM) and phenolicglycosides ( PG ; 1 5-35 g/kg DM) were higher in willow trees compared to their concentrations (CT ; 2-3 g/kg DM) and (PG; 2-9 g/kg OM) in control drought pastures. Experiments involving short drought pasture, long drought pasture and willow fodder blocks as treatment groups were grazed by ewes for 10 weeks in regular breaks from mid February to early May. Ewes were mated during this period and were joined together after mating and grazed on normal pasture until weaning. Live weight (LW) change and body condition score (BCS) were recorded throughout the experiments, whilst reproductive performance of ewes was measured as the number of lambs recorded at ultrasound pregnancy scanning, lambing, docking and weaning. Measurements on wool production were also recorded at weaning. In 2003, experimental ewes grazed control drought pastures (short and long) and willow fodder blocks (restricted and full access) as treatment groups (n= 1 00 ewes/group; Chapter 2). Ewes grazing short drought pasture had an allowance of 0.8 k g DM/ewe/d whilst ewes with restricted access had an allowance of 0.8 kg DM/ewe/d from drought pasture and 0 .4 kg OM/ewe/d from willow fodder blocks. Ewes in full access treatment group had no access to pasture but were confined to willow fodder blocks at an allowance of 2.0 kg OM/ewe/d, which was the same allowance given to long drought pasture ewes. Ewes grazing short drought pasture lost weight at approximately 1 00g/d and recorded a low reproductive rate (90 lambs weaned/100 ewes mated) with a high proportion of single lamb births. Live weight loss was significantly reduced to 40 g/d in ewes grazing willow fodder blocks (full access) with a 20% units increase in reproductive rate due to more multiple births (P <0. 05). Ewes grazing long drought pasture performed intermediate to ewes with full access to fodder blocks and ewes grazing short drought pasture, whilst ewes with restricted access performed similar to ewes grazing short drought pasture. In 2004 (Chapter 3), the restricted access to willow fodder blocks treatment was eliminated from the study and the number of ewes was increased to 165 ewes per treatment group. Performance of ewes grazing short drought pasture was similar to that of ewes grazing short drought pasture in 2003 , with ewes loosing live weight (40g/d) and a low reproductive rate (90 lambs weaned/l00 ewes mated) whilst ewes grazing long drought pasture gained L W (54 g/d) and had a higher reproductive rate (P<0.05). Ewes grazing willow fodder blocks performed better than ewes grazing short drought pasture by maintaining L W and their reproductive rate was intermediate to ewes grazing short and long drought pasture. In 2005, a short grazing trial with rumen fistulated sheep was conducted to study the effect of supplementing willow to ewes grazing drought pastures upon plasma amino acid concentrations (Chapter 4) and upon the microbiology of the rumen (Chapter 5 and 6). Grazing occurred during summer/autumn for 10 weeks with two treatment groups; control (short drought pasture; n=7) at an allowance of 0.8 kg DM/ewe/d and ewes grazing short drought pasture at 0.8 kg DM/ewe/d plus a supplement of fresh willow at 1.4 kg fresh willow/ewe/d (n=7) . Blood samples for the quantification of plasma amino acids were collected at week 5 and 10, with L W and BCS measured at fortnightly intervals. Short drought pasture in this experiment had a low pasture mass (2000 k g DM/ha) and a low nutritive value (8 MJ/kg DM), whilst willow had a higher M E of 10 MJ/kg OM. Both groups of ewes lost live weight at the rate of 50 g/d. Plasma concentration of 3 methylhistidine (3-MTH; 88 vs 127μ mole/L) at week 5 and non essential amino acids (NEAA; 1082 vs 1417μ mo1e/L) at week 5 and ( 1155 vs 1324 μ mole/L) at week 10, were substantially lower (P<0 .05) in w illow supplemented ewes than control ewes. It was concluded that the increased reproductive rate from willow supplementation in ewes grazing drought pasture might be partly explained by reduced body protein catabolism, besides also increasing plasma branched chain amino acids CBCAA) and essential amino acids (EAA) concentrations. To investigate the effects of willow supplementation on rumen microbes, rumen samples were collected during the 2005 experiment with fistulated ewes over a 10 week period. The study involved the use of a molecular technique ( Chapter 5), denaturing gradient gel electrophoresis (DGGE), to compare the rumen microbial populations between the control and supplemented ewes and a cultivation technique (Chapter 6) to study the effect on rumen bacteria of ewes grazing drought pastures with and with out willow supplementation. DGGE analysis of the V3 region of 16S ribosomal RNA genes in DNA extracted from samples of rumen contents taken fortnightly over a 10 week feeding period showed a distinct difference in banding patterns between treatment groups which progressively developed over time, showing rumen microbial adaptation to willow supplementation. However, phylogenetic analysis of the DNA sequences retrieved from the DGGE bands from willow-supplemented and control ewes did not cluster by treatment group. It was deduced that willow supplementation induced a change in rumen bacterial populations through selecting sub-populations of organisms already present in the rumen. The changes in the rumen bacterial populations is attributed to the ability of these bacteria to metabolise secondary compounds in willow such as phenolicglycosides and flavanoid monomers and their ability to resist the inhibitory effects of condensed tannins. The cultivation study involved enumeration, isolation and purification of bacterial colonies on Complete Carbohydrate, Salicin, Xylan, Cellulose and Willow media followed by full characterisation of a representative set of pure bacterial cultures. Total bacterial counts on the above media at week 5 and week 10 were generally lower in willow-supplemented ewes compared to control ewes and the 16S rRNA gene sequences of the majority of iso lates characterised from both Salicin and Xylan media, were most closely related to species from the Pseudobutyrivibrio genus. Isolates from Willow medium clustered as two distinct groups. One group (mostly isolated from control ewes) was made up of mainly of organisms not usually associated with the rumen and probably represent non-resident organisms that are passing through the rumen. The other group of bacteria were mainly retrieved from willow-supplemented ewes and were most closely related to species of the Ofsenella genus. Compared to bacteria isolated on Salicin and Xylan media, isolates on Willow medium showed little ability to ferment various carbohydrates or trypticase (hydrolysed protein) but were able to utilise secondary compounds from willow. It was concluded that willow fodder blocks are useful sources of supplementary fodder for mating ewes during drought situations. Both the field and m icrobiological studies showed adaptation to the willow supplementary diet, including the detection of Olsenelfa-like bacteria for the first time in the rumen. It is suggested that the principal purpose of the rumen investigation is the degradation of secondary compounds present in willow.