Made available in DSpace on 2014-10-09T12:23:57Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:46Z (GMT). No. of bitstreams: 1 01107.pdf: 2937024 bytes, checksum: 79731d7cd4026a178cbdc41ff721c163 (MD5) / Dissertacao (Mestrado) / IEA/D / Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo - FMVZ/USP

Orientador: Alexandre Xavier Falcão / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T18:50:47Z (GMT). No. of bitstreams: 1 Gomes_JancarloFerreira_D.pdf: 2024129 bytes, checksum: eeb77aec9d0bc9546a0141b794a17319 (MD5) Previous issue date: 2008 / Resumo: No presente trabalho, averiguamos sobre processamentos de amostra fecal, desenvolvimento e avaliação de um sistema de computador para análise de imagem parasitária. Observamos que a etapa de processamento de fezes consistiu a etapa crítica, mas, essencial para o fornecimento de estruturas parasitárias limpas, com reduzida quantidade de microimpurezas fecais, ao sistema de análise por computador. Algumas técnicas parasitológicas convencionais, utilizando ou não kits comerciais, foram estudadas, e a de TF-Test (Three Fecal Test) apresentou menor teor de microimpurezas no final de processamento fecal. Todavia, este teor de microimpurezas era ainda muito grande, sendo ainda inadequado para análise computacional. Modificações foram introduzidas à técnica de TF-Test, com coloração dos componentes do sedimento fecal, utilizando um corante desenvolvido à base de Lugol, e seguido por degradação alcalina de microimpurezas, com uma solução clarificadora, previamente padronizada. Assim, a técnica de TF-Test Modificada contribuiu para o fornecimento de parasitos com poucas microimpurezas. O desenho do protótipo do sistema computacional para a análise de imagens incorporou: a técnica de TFTest Modificada; um microscópio óptico adaptado a uma bomba sucção peristáltica para conduzir uma alíquota de suspensão fecal processada à uma lâmina ou câmara tubular, onde estruturas parasitárias ou não apareciam em imagem; uma câmera digital; um monitor de vídeo para regulagem e captura de imagens; e um computador. Um banco de imagens foi construído, após coletas de amostras fecais foram efetuadas previamente em 4 regiões diferentes (Campinas, Botucatu, Avaré e Piraju) do estado de São Paulo, onde as enteroparasitoses são prevalentes. Os procedimentos de biossegurança e controle de qualidade contribuíram para que a perda da amostra fecal fosse pequena, não ultrapassando de 8%. Foi obtido um total de 16 espécies parasitárias, constituídos de helmintos e protozoários, e estes proveram uma coleção de 1.126 imagens ao computador. Ademais, o banco de imagens foi formado por informações adquiridas de 5.626 componentes parasitários e não parasitários, assim como, de dados sobre suas características de forma, textura e cor. A análise computacional baseou-se em um sistema de pipeline de técnicas de processamento de imagens, incluindo o uso de uma técnica denominada de Image-Foresting Transform (IFT). O pipeline consistiu em técnicas de segmentação de imagens para separar estruturas parasitárias e impurezas do fundo das imagens; em técnicas de extração para codificar características da forma, da cor e da textura dos parasitos; e em técnicas de reconhecimento e delineamento de imagens, visando distinguir parasitos de microimpurezas, de acordo com suas características próprias. A técnica de análise de imagens por computador (CIA) foi avaliada em comparação com a técnica de TF-Test Modificada de microscopia óptica, demonstrando alta sensibilidade de 95,3%, especificidade de 96,4%, e eficiência de 96,2 %. O conceito de concordância observada entre as duas técnicas estudadas foi de Quase Perfeito, em virtude do índice kapa (k) ter sido elevado, de 0,88. Esta técnica demonstrou ser altamente reprodutível, quando se ensaiaram em 10 diferentes ocasiões. Os achados deste trabalho apresentam perspectivas para industrialização do protótipo aqui desenvolvido, causando impacto na área de Saúde Pública, pois, no exame de fezes para população, há uma forte demanda de um sistema de automatização para detecção de enteroparasitos / Abstract: In the present study, we investigated on fecal sample processings, development and evaluation of a computer system for parasite image analysis. We observed that the fecal processing step was critical, but, essential for providing clean parasite structures, with reduced amount of fecal microdebris, to the system of computer analysis. Several conventional parasitologic techniques, using or not commercial kit, were studied, and TF-Test (Three Fecal Test) showed lower rate of microdebris at the end of fecal processing. However, this microdebris rate was still great, being unsuitable for computer analysis. The TF-Test technique was modified by staining the fecal sediment components, with a Lugol-based stain, followed by an alkaline degradation of fecal microdebris, using a clarifyer solution. So, the modified TF-Test technique became capable to supply parasite structure with little fecal microdebris. The prototype design of the computer system for the image analysis incorporated: the modified TF-Test technique; a optical microscope coupled to a peristaltic suction pump for leading an aliquot of processed fecal suspension to the tubular slide or chamber, where the parasite structures or microdebris appeared in tridimensional images; a digital camera; a video monitor to calibrate and capture images; and a computer. An image database was formed, after collecting fecal samples from prevalent regions (Campinas, Botucatu, Avaré and Piraju) of the State of São Paulo for enteroparasitosis. The total fecal sample loss was low, being less than 8%, since biosecurity and laboratory quality control protocols were frequently checked. A total of 16 parasite species were identified, consisting of helminths and protozoans, which provided a collection of 1.126 parasite images to the computer. Moreover, the image database was formed by information acquired from 5.626 parasite and nonparasite components, in addition to data on their shape characteristics, texture and color. The computational analysis was based on a pipeline of image processing techniques, including the use of a technique known as Image-Foresting Transform (IFT). The pipeline consisted of: a technique for image segmentation, in order to separate parasites and microdebris from background image; a technique for feature extraction to encode shape, color and texture characteristics of parasites; and techniques for pattern recognition and delineation, permitting to distinguish parasites from microdebris, according to their own features. The technique of computer image analysis (CIA) was evaluated in comparison with the optical microscope technique, named modified TF-Test, demonstrating a high sensitivity of 95,3%, specificity of 96,4% and efficiency of 96.2%. The agreement between two techniques was ranked as Almost Perfect, since the kappa (k) index has been high as much as 0.88. This technique proved to be reproducible, in a study, in which the assay was repeated 10 times, in different occasions. Our findings present good perspectives for the industrial production of the here developed prototype, causing impact on the Public Health area, since, in the fecal examination of the population, there is a strong demand for an automated system of enteroparasite detections / Doutorado / Doutor em Parasitologia

Reduced availability of high energy-content prey (nutritional stress) is a predominant hypothesis to explain the decline of Steller sea lion (Eumetopias jubatus) populations in western Alaska from the late 1970' s to the late 1990' s. Animals may physiologically respond to consuming insufficient prey by increasing stress levels and decreasing metabolic rates. It may thus be possible to identify nutritional stress by measuring concentrations of glucocorticoids (stress) and thyroid hormones (metabolism) shed in feces. However, techniques to measure thyroid hormone concentrations from Steller sea lion feces have not been developed. We quantified variation of triiodothyronine (T3) and thyroxine (T4) concentrations in Steller sea lion feces following injections of thyrotropin (TSH) into four captive animals. Glucocorticoids (GC) were also assayed to examine any relationship to stimulated thyroid hormone secretion. We found that fecal T3 peaked 48 h post-injection and increased 25-57% in three sea lions (all animals, p=0.03). Pre-injection GC increases indicated stress from isolation for baseline fecal collections, but post-injection increases could not be confirmed as a response to TSH injections or as a product of the study design. The results demonstrated that pre- and post-injection changes in fecal GC and T3 concentrations were consistent with predictions of an increased stress response and metabolic rate within the animals. We then measured T3 and GC concentrations in 834 Steller sea lion fecal samples collected in 2005 and 2006 from 15 sites (haulouts and rookeries) between British Columbia and the Central Aleutian Islands. Overall, GC concentrations did not differ between haulout populations (western 2006 pre-pupping and eastern 2005 post-pupping). Fecal hard-part analyses revealed a lower energy-content diet in the western population, suggesting that diet quality is a relevant hypothesis to explain slightly higher GC concentrations found in the western population, specifically the Aleutian Islands region. However, nutritional stress could not be substantiated through T3 concentrations. The rookeries possessed the highest energy-content diets, but also exhibited a nutritional stress response with a significantly higher GC and lower T3 concentration than either haulout population (possibly related to lactation or decreased foraging opportunities), but T3 comparisons performed at scales of site and region were inconclusive. / Science, Faculty of / Zoology, Department of / Graduate

Please read the abstract in the 00front part (pp12-17) of this document / Dissertation (MSc (Medical Virology))--University of Pretoria, 2004. / Medical Virology / unrestricted

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The global climate is strongly regulated by the oceans, which store carbon away from the atmosphere for long periods. In an effort to understand the role of the oceans in the carbon cycle, it is necessary to understand the nuances of specific regional and functional marine ecosystems. The continental shelf of the West Antarctic Peninsula (WAP) is one particularly important regional ecosystem that plays a vital role in the Southern Ocean carbon export. Within the seasonally productive marginal ice zone of the WAP, I sought to identify the long-term drivers of particulate organic carbon (POC) flux. The vast majority of exported POC on the WAP was previously found to be made up of krill fecal pellets. I provide evidence that supports the hypothesis that the inherent life cycle of krill drives the observed 5-year oscillation in POC export. At the end of their life cycle, when krill are at their largest body size, the WAP experiences anomalously high POC export events through the production and sinking of large, carbon-rich krill fecal pellets. Conversely, when krill are young and small, POC export is anomalously low. This pattern shows that ecology exerts a first-order control on the the biogeochemical cycles of the WAP. Upon identifying the source and driver of POC export on the WAP, I set out to determine the role heterotrophic bacteria play in POC flux attenuation. I collected krill fecal pellets on the WAP over three years and measured bacterial metabolic activity in terms of bacterial production and respiration, thereby identifying the amount of organic carbon within the sinking fecal pellets that is lost due to bacteria. Overall, fecal pellet POC turnover rate by bacteria is very low. The relationship between bacteria and POC is complex with each having an affect on the other. Despite varied reactions of the free-living bacterial populations to the presence of krill fecal pellets, a consistent pattern emerged in the concentration of nucleic acid within each bacterial cell. Access to fecal pellets increased the metabolic activity of the free-living bacterial population. This finding shows that the egestion of krill fecal pellets metabolically stimulates the surrounding bacterial community, even though bacteria play a minor role in fecal pellet POC flux attenuation. Though bacteria were found to play a minimal role in organic carbon uptake on krill fecal pellets, they are still vital members of the WAP ecosystem and biological pump. I next sought to identify which bacteria in particular were responsible for colonizing and consuming the fecal pellet POC. Krill fecal pellets were genetically sequenced after timed exposure to the free-living water column bacterial community. I found that there is an endemic population of bacteria that are associated with each population of krill and their fecal pellets. This community of fecal pellet-associated bacteria does not change over time, indicating little colonization by free-living bacteria. Krill fecal pellets, aside from being good agents of POC export, seem to be selective environments for certain specialized copiotrophic bacteria. Further, I find that only a small subset of these endemic copiotrophs actively partake in carbon consumption on krill fecal pellets. Overall, these results show that a small endemic, specialized bacterial community play an outsized role in krill fecal pellet POC degradation and flux attenuation, but that krill fecal pellets remain efficient agents of carbon export to the deep ocean.

The glycerol enol ether, fecapentaene-12, is a direct acting fecal mutagen that is formed in the lower portion of the gastrointestional tract by anaerobic bacteria. The biological precursor to fecapentaene-12 is a natural product of mammalian origin whose role in the etiology of colon cancer is unknown. Preliminary evidence indicated that the precursor may be a plasmalogen with an intact pentaenol ether moiety. Further structural studies by means of degradative methods and chromatographic techniques enabled the structure of the precursor to be elucidated. Based on the structure of the precursor, the name plasmalopentaene-12 was coined. Synthetic methodology was developed for obtaining synthetic plasmalopentaene12. This was necessary in order to confirm the structure and to determine the precursor's biological role. The synthetic methodology proceeded through a novel "acyl migration" which enables the highly labile pentaenol ether to be generated late in the synthesis. Model studies indicated that this was a feasible pathway. It was also determined that this methodology may be highly adaptable to the synthesis of other plasmalogens and may also provide a new synthetic route to fecapentaene-12. / Ph. D.

Master of Science / Department of Diagnostic Medicine/Pathobiology / T. G. Nagaraja / Shiga toxin-producing E. coli O157 is a major foodborne pathogen. The organism colonizes the hindgut of cattle and is shed in the feces, which serves as a source of contamination of food. Generally, cattle shed E. coli O157 at low concentrations (≤ 10[superscript]2 CFU/g), but a subset of cattle, known as “super-shedders”, shed high concentrations (>10[superscript]3 CFU/g) and are responsible for increased transmission between animals and subsequent hide and carcass contamination. Therefore, concentration data are an important component of quantitative microbial risk assessment. A four-plex quantitative PCR (mqPCR) targeting rfbE[subscript]O157, stx1, stx2 and eae was developed and validated to detect and quantify E. coli O157 in cattle feces. Additionally, the applicability of the assay to detect E. coli O157 was compared to conventional PCR (cPCR) targeting the same four genes, and a culture method. Specificity of the assay to differentially detect the four genes was confirmed. In cattle feces spiked with pure cultures, detection limits were 2.8 x 10[superscript]4 and 2.8 x 10[superscript]0 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. Of the 100 samples that were randomly picked from the 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from the 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar’s chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. Applicability of the assay to quantify E. coli O157 was determined with feedlot cattle fecal samples (n=576) and compared to spiral plate method. Fecal samples that were quantifiable for O157 by mqPCR (62/576; 10.8%) were at concentrations of ≥ 10[superscript]4 CFU/g of feces. Only 4.5% (26/576) of samples were positive by spiral plate method, with the majority (17/26; 65.4%) at below 10[superscript]3 CFU/g. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect and quantify E. coli O157 in cattle feces.

White feces syndrome (WFS) is an emerging problem for penaeid shrimp farming industries in SE Asia countries, Thailand, Malaysia, Vietnam, Indonesia, China, and in India. This occurrence of this syndrome is usually first evidenced by the appearance of white fecal strings floating on surface of the shrimp ponds. The gross signs of affected shrimp include the appearance of a whitish hindgut and loose carapace, and it is associated with reduced feeding and growth retardation. To investigate the nature of the white feces syndrome, samples of white feces and shrimp hepatopancreas tissue were collected from Penaeus vannamei in affected farms in Indonesia, and these were examined histologically. Within the white feces, we found densely packed spores of the microsporidian Enterocytozoon hepatopenaei (abbreviated as EHP) and relatively fewer numbers of rod-shaped bacteria. From WFS ponds, hepatopancreas samples form 30 individual shrimp were analyzed by histology and in situ hybridization. The results showed that all of the shrimp examined were infected with EHP accompanied by septic hepatopancreatic necrosis (SHPN). Midgut epithelial cells were also infected and this increased the number of tissue types being affected by EHP. By PCR, EHP was detected in all the samples analyzed from WFS-affected ponds, but not in those sampled from healthy shrimp ponds. To determine the modes of transmission for this parasite, we performed feeding and cohabitation bioassays, the results showed that EHP can be transmitted through per os feeding of EHP-infected hepatopancreas tissue to healthy shrimp and through cohabitation ofinfected and healthy shrimp. In addition, we found the use of Fumagillin-B, an antimicrobial agent, was ineffective in either reducing or eliminating EHP in infected shrimp.