M.Sc. (Botany) / Please refer to full text to view abstract

Fotossensibilização refere-se ao aumento da resposta à radiação ultravioleta da luz solar, causada por um agente fotodinâmico na pele. A sensibilidade à fotossensibilização de 80 cordeiros mestiços desmamados e manejados em lotação rotativa, durante 120 dias, foi avaliada em pastagem de Brachiaria brizantha cv. Marandu. Os cordeiros foram suplementados a 1% do seu peso corporal com os tratamentos, a saber: A - suplemento basal com 12% PB; B - suplemento com 18% de PB; C - suplemento com 18% de PB + propionato de Zn e D - suplemento com 18% de PB + cloreto de colina protegida. Ao término do experimento os animais foram abatidos. Os objetivos foram: avaliar o efeito da adaptação dos animais suplementados às condições de manejo em Brachiaria brizantha e a toxidez da planta aos cordeiros; caracterizar as causas da fotossensibilização; viabilizar o possível controle da verminose associada ao manejo de cordeiros em lotação rotativa e à suplementação mineral e proteica, com ou sem adição de aditivos; relacionar os teores de saponina e a presença de esporos de Pithomyces chartarum em amostras da forragem à ocorrência de casos de fotossensibilização hepatógena. O delineamento experimental foi em blocos completos e casualizados, com quatro tratamentos e quatro repetições, num total de 5 animais por tratamento. Realizou-se a análise da variância para as variáveis e as médias foram comparadas pelo teste t-Student ao nível de significância de 5%. Foram observadas baixas contagens de esporos de Pithomyces chartarum (0 - 15.000 esporos) na parte senescente da planta. A concentração de saponina protodioscina variou de 0,52 a 0,65%. Do 8º ao 58º dia ocorreram 20% de mortalidade dos animais. A avaliação histopatológica, do fígado dos animais que vieram a óbito, foi compatível com as lesões causadas pela saponina. Os padrões histológicos das lesões hepáticas encontradas nos animais, ao final do experimento, não diferiram entre os tratamentos, bem como para as análises bioquímicas: colesterol, triglicerídeos, bilirrubinas, proteína total, albumina, creatinina, ureia, AST, GGT. Ocorreram diferenças nas concentrações séricas de bilirrubinas, proteínas, albumina, AST, GGT, ureia, creatinina, colesterol, triglicerídeos, entre cordeiros sobreviventes e os que morreram. O método rotativo foi efetivo no controle da verminose, ficando a contagem de ovos por grama de fezes (OPG) abaixo de 500 ao final do experimento, não ocorrendo diferença entre os suplementos proteicos e entre os aditivos utilizados. Não houve efeito no desempenho dos cordeiros, que chegaram ao final do experimento, pela inclusão de suplementos proteicos e de aditivos. / Photosensitization refers to increase in response to ultraviolet radiation from sunlight, caused by a photodynamic agent into the skin. The sensitivity of the photosensitization at 80 crossbred lambs weaned was evaluated on Brachiaria brizantha cv. Marandu managed in rotational grazing for 120 days. Lambs were supplemented to 1% of their body weight with different treatments: A - basal supplement with 12% CP; B - supplement with 18% CP; C - supplement with18% CP + zinc proprionate and D - supplement with18 % of CP + protected choline chloride. At the end of the experiment the animals were slaughtered. The objectives were to evaluate the effect of adaptation of animals supplemented to handling Brachiaria brizantha and toxicity of the plant to lambs weaned; to characterize the causes of photosensitivity, the possible control of parasitism associated with the management of sheep grazing on rotational grazing, mineral supplementation and protein with or without the addition of additives; to relate the levels of saponin and the presence of spores Pithomyces chartarum in samples of forage and the occurrence of hepatic photosensitization. The experimental design was a complete randomized block design and assigned to four treatments and four replications for a total of five animals per treatment. The analysis of variance was performed and the means test t-Student a significance level of 5% for the effect of each treatment was used. Low count of Pithomyces chartarum spores (spores 0-15,000) in the senescent part of the plant was observed. The concentration of saponin protodioscine ranged from 0.52 to 0.65 %. Occurred between the 8º and 58º day mortality of 20 % of the animals. Histopathology of the liver of animals that died during the experiment was consistent with lesions determined by saponin. The histological patterns of liver lesions found in animals at the end of the experiment did not differ between treatments, as well as, for biochemical analysis: cholesterol, triglycerides, bilirubin, total protein, albumin, creatinine, urea, AST and GGT. There were differences in serum bilirubin, protein, albumin, AST, GGT, urea, creatinine, cholesterol and triglycerides between survivors and that died. The rotational grazing system was effective in the control of nematode parasites getting below 500 EPG at the end of the experiment, there was no difference between the protein concentrations, nor for the additives used. The additives zinc oxide and choline chloride did not improve the performance of sheep.

Fotossensibilização refere-se ao aumento da resposta à radiação ultravioleta da luz solar, causada por um agente fotodinâmico na pele. A sensibilidade à fotossensibilização de 80 cordeiros mestiços desmamados e manejados em lotação rotativa, durante 120 dias, foi avaliada em pastagem de Brachiaria brizantha cv. Marandu. Os cordeiros foram suplementados a 1% do seu peso corporal com os tratamentos, a saber: A - suplemento basal com 12% PB; B - suplemento com 18% de PB; C - suplemento com 18% de PB + propionato de Zn e D - suplemento com 18% de PB + cloreto de colina protegida. Ao término do experimento os animais foram abatidos. Os objetivos foram: avaliar o efeito da adaptação dos animais suplementados às condições de manejo em Brachiaria brizantha e a toxidez da planta aos cordeiros; caracterizar as causas da fotossensibilização; viabilizar o possível controle da verminose associada ao manejo de cordeiros em lotação rotativa e à suplementação mineral e proteica, com ou sem adição de aditivos; relacionar os teores de saponina e a presença de esporos de Pithomyces chartarum em amostras da forragem à ocorrência de casos de fotossensibilização hepatógena. O delineamento experimental foi em blocos completos e casualizados, com quatro tratamentos e quatro repetições, num total de 5 animais por tratamento. Realizou-se a análise da variância para as variáveis e as médias foram comparadas pelo teste t-Student ao nível de significância de 5%. Foram observadas baixas contagens de esporos de Pithomyces chartarum (0 - 15.000 esporos) na parte senescente da planta. A concentração de saponina protodioscina variou de 0,52 a 0,65%. Do 8º ao 58º dia ocorreram 20% de mortalidade dos animais. A avaliação histopatológica, do fígado dos animais que vieram a óbito, foi compatível com as lesões causadas pela saponina. Os padrões histológicos das lesões hepáticas encontradas nos animais, ao final do experimento, não diferiram entre os tratamentos, bem como para as análises bioquímicas: colesterol, triglicerídeos, bilirrubinas, proteína total, albumina, creatinina, ureia, AST, GGT. Ocorreram diferenças nas concentrações séricas de bilirrubinas, proteínas, albumina, AST, GGT, ureia, creatinina, colesterol, triglicerídeos, entre cordeiros sobreviventes e os que morreram. O método rotativo foi efetivo no controle da verminose, ficando a contagem de ovos por grama de fezes (OPG) abaixo de 500 ao final do experimento, não ocorrendo diferença entre os suplementos proteicos e entre os aditivos utilizados. Não houve efeito no desempenho dos cordeiros, que chegaram ao final do experimento, pela inclusão de suplementos proteicos e de aditivos. / Photosensitization refers to increase in response to ultraviolet radiation from sunlight, caused by a photodynamic agent into the skin. The sensitivity of the photosensitization at 80 crossbred lambs weaned was evaluated on Brachiaria brizantha cv. Marandu managed in rotational grazing for 120 days. Lambs were supplemented to 1% of their body weight with different treatments: A - basal supplement with 12% CP; B - supplement with 18% CP; C - supplement with18% CP + zinc proprionate and D - supplement with18 % of CP + protected choline chloride. At the end of the experiment the animals were slaughtered. The objectives were to evaluate the effect of adaptation of animals supplemented to handling Brachiaria brizantha and toxicity of the plant to lambs weaned; to characterize the causes of photosensitivity, the possible control of parasitism associated with the management of sheep grazing on rotational grazing, mineral supplementation and protein with or without the addition of additives; to relate the levels of saponin and the presence of spores Pithomyces chartarum in samples of forage and the occurrence of hepatic photosensitization. The experimental design was a complete randomized block design and assigned to four treatments and four replications for a total of five animals per treatment. The analysis of variance was performed and the means test t-Student a significance level of 5% for the effect of each treatment was used. Low count of Pithomyces chartarum spores (spores 0-15,000) in the senescent part of the plant was observed. The concentration of saponin protodioscine ranged from 0.52 to 0.65 %. Occurred between the 8º and 58º day mortality of 20 % of the animals. Histopathology of the liver of animals that died during the experiment was consistent with lesions determined by saponin. The histological patterns of liver lesions found in animals at the end of the experiment did not differ between treatments, as well as, for biochemical analysis: cholesterol, triglycerides, bilirubin, total protein, albumin, creatinine, urea, AST and GGT. There were differences in serum bilirubin, protein, albumin, AST, GGT, urea, creatinine, cholesterol and triglycerides between survivors and that died. The rotational grazing system was effective in the control of nematode parasites getting below 500 EPG at the end of the experiment, there was no difference between the protein concentrations, nor for the additives used. The additives zinc oxide and choline chloride did not improve the performance of sheep.

Facial eczema disease (FE) is a secondary photosensitisation disease of ruminants caused by exposure to the mycotoxin sporidesmin. Resistance to FE has a significant genetic component and previous research has included a whole genome scan and investigation of candidate genes. The aim of this study was to use multiple approaches to identify genes associated with resistance to FE. ABC transporters have been considered as putative candidate genes for FE since the yeast ABC transporter, PDR5, was found to modulate sensitivity to sporidesmin in Saccharomyces cerevisiae. A previous study had shown that hepatic expression of the ovine ABC transporter, ABCB1, was induced following exposure to sporidesmin but only in resistant animals (Longley (1998) PhD Thesis, University of Otago). In the present study, using qRT-PCR, a difference in the expression of ABCB1 between resistant and susceptible animals was not confirmed. It is concluded that ABCB1 is not likely to be a candidate gene for FE. As the full genome sequences for several mammalian species are now available, phylogenetic analyses were used to identify the most likely mammalian ortholog of the yeast PDR5 protein. This analysis found that the yeast PDR5 protein was most closely related to the mammalian ABCG sub-family. The human ABCG sub-family has five members one of which, ABCG2, is a known xenobiotic transporter. Comparative mapping of ABCG2 indicated that it co-localised to a region of the sheep genome weakly associated with resistance to FE. The full-length sequence of ovine ABCG2 was determined and two synonymous polymorphisms were found. These two polymorphisms, together with an intronic SNP were genotyped across a panel of selection-line animals. The allele frequencies of the intronic SNP were found to be significantly different between the selection lines, providing evidence for the association of ABCG2 with resistance to FE. The hepatic expression of ABCG2 was examined but no differential expression between the selection-lines was observed. Global gene expression profiling via microarray analysis was undertaken as a novel approach to identify candidate genes. Differences in gene expression were examined between naïve and sporidesmin-dosed resistant and susceptible animals using a bovine cDNA microarray. A small number of differentially expressed genes were identified. Follow-up studies found that there were a relatively high number of errors in EST identity. Eight differentially expressed genes were selected for confirmation by Northern analysis. Six of these genes were shown to be differentially expressed, but neither the patterns nor the magnitude of the differential expression reflected that observed on the microarray. One of the six genes identified as differentially expressed was catalase, which has previously been implicated in resistance to FE. This finding validates the approach taken using gene expression profiling to identify candidate genes. The final approach used in this study necessitated the development and characterisation of an in vitro system for studying sporidesmin toxicity. The system chosen was a human hepatoma cell line, HepG2. To date the only effective treatment for FE is the prophylactic administration of high levels of zinc sulphate. The mechanism of protection by zinc is unknown, but zinc is known to be a potent modulator of gene expression. Conceptually, any genes modulated by zinc are possible candidates for resistance to FE. It was shown that zinc pre-treatment could protect HepG2 cells against sporidesmin-induced cytotoxicity. Equivalent protection was provided by the addition of zinc in the presence of the transcriptional inhibitor actinomycin D, suggesting that the mechanism of zinc protection is independent of de novo gene transcription. Overall, the goal of this project was to find genes to assist selection of sheep resistant to FE. Toward this goal, this research has identified several new candidate genes and avenues for investigation.

Made available in DSpace on 2016-04-28T20:16:32Z (GMT). No. of bitstreams: 1 2009 - Josilene Nascimento Seixas.pdf: 4044215 bytes, checksum: dfe1223a8c9106028077731ca7dd2fe5 (MD5) Previous issue date: 2009-02-18 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Numerous outbreaks of photosensitization in herbivorous animals maintained on Brachiaria pastures occur in Brazil, and the economic losses caused are of great concern due to the large areas in the country planted with this grass. The vast majority of outbreaks has been attributed to the sporidesmin containing spores of Pithomyces chartarum, a fungus which occurs in many countries of temperate climate. However, there are differences between the liver lesions in animals that develop photosensitization on pastures of Brachiaria spp and the ones described in P. chartarum poisoning. In several outbreaks reported from Brazil, no evidence for toxicity of the spores was revealed. As the isolation of toxic saponins from Brachiaria grasses has been reported in the literature, the real cause of the photosensitization in Brazil needs to be clarified, in order to be able to adopt correct preventive measures to avoid the condition. The main objective of this study was to show the different epidemiological, clinical, pathological and toxicological aspects of the two poisonings, based on data from the literature and our own observations of pithomycotoxicosis in Portugal and on the data of the photosensitization which occurs on Brachiaria pastures in Brazil. We concluded that the saponin content of the grass is be responsible for the outbreaks of photosensitizing disease which occur annually on Brazilian pastures. / Numerosos surtos de fotossensibiliza??o em animais mantidos em pastos de Brachiaria spp v?m ocorrendo no Brasil e as perdas econ?micas resultantes t?m sido foco de preocupa??o, em especial devido ?s grandes ?reas cultivadas dessa gram?nea no pa?s. A maioria dessas ocorr?ncias vem sendo atribu?das ? esporidesmina, presente em esporos do fungo Pithomyces chartarum, que ocorre em muitos pa?ses de clima temperado. No entanto, h? diferen?as entre o aspecto an?tomo-histopatol?gico verificado no f?gado de animais que desenvolvem fotossensibiliza??o em pastos de Brachiaria spp e o descrito na intoxica??o por P. chartarum. Nos diversos surtos relatados no Brasil n?o se conseguiu demonstrar a toxidez dos esporos. Como o isolamento de saponinas t?xicas tem sido reportado em pastagens de Brachiaria, a causa dos surtos de fotossensibiliza??o no Brasil precisa ser esclarecida, para que corretas medidas preventivas sejam adotadas e, a doen?a evitada. Este trabalho tem por objetivo principal demonstrar, atrav?s das diferen?as epidemiol?gicas, cl?nico-patol?gicas e toxicol?gicas peculiares ?s duas condi??es, colhidas na literatura e confrontadas com nossas pr?prias observa??es sobre pitomicotoxicose em Portugal e intoxica??o pela Brachiaria sp no Brasil, que as saponinas contidas na Brachiaria sp s?o respons?veis pelos surtos de fotossensibiliza??o que ocorrem anualmente nas pastagens brasileiras.