Orientador: Antonio Nader Filho / Banca: Hélio Langoni / Banca: Nilson Roberti Benites / Banca: Humberto Tonhati / Banca: Luiz Augusto do Amaral / Resumo: Os exames de palpação, inspeção e teste da caneca de fundo escuro, bem como o California Mastitis Test (CMT) foram realizados em 735 quartos mamários de búfalas leiteiras pesquisa de alterações clínicas e subclínicas nos quartos e em suas respectivas secreções. Independentemente dos resultados encontrados, foram coletadas amostras de leite para análises microbiológicas e de contagem de células somáticas (CCS). O isolamento microbiológico foi realizado em ágar sangue de ovino 5% e em ágar MacConkey, com 72 horas de incubação em aerobiose a 37oC. Foram procedidas, a cada 24 horas, leitura e identificação dos isolamentos. A ocorrência de quadros de mastite clínica foi representada por um único caso (0,14%) entre os 735 quartos mamários analisados, ocorrendo na fase inicial da lactação e envolvendo microrganismos de origem ambiental (Morganella morganii, Enterobacter aerogenes). Foram considerados quadros de mastite subclínica aqueles com resultados positivos ao CMT acompanhados de exame microbiológico positivo. A freqüência observada de quadros de mastite subclínica entre os 734 quartos mamários estudados foi de 20,03%, dos quais isolou-se principalmente microrganismos contagiosos. Os resultados negativos ao CMT predominaram durante todo o estudo (69,07%), independente da fase de lactação ou estação do ano consideradas, tendo sido observados resultados positivos do exame microbiológico em 57,58% das amostras CMT negativas. Foi elevada a freqüência de microrganismos classificados como contagiosos entre as amostras de leite analisadas, independente da estação do ano e da fase de lactação, entre os quais prevaleceu o Corynebacterium spp. (47,67%), entre os 579 microrganismos pertencentes a 19 gêneros bacterianos crescidos em cultura pura ou em associação. Considerando-se a falta de homogeneidade entre as variâncias, as contagens de células... (Resumo completo, clicar acesso eletrônicio abaixo) / Abstract: A total of 735 mammary quarters from buffalo cows were submitted to inspection, palpation, strip cup test and California Mastitis Test (CMT) in order to diagnose clinical and subclinical mastitis. Independent of the observed results, milk samples were collected from each mammary quarter to microbiological exams and automatic somatic cell counts (SSC). Milk samples were inoculated in 5% ovine blood agar and MacConkey agar and incubated for 72 hours under 37oC and aerobic condition. Growth observations and identifications were performed every 24 hours. One (0,14%) out of 735 mammary quarters studied presented clinical mastitis. It occurred in first stage of lactation (0 to 60th day post calving) and environmental microorganisms were isolated (Morganella morganii, Enterobacter aerogenes). Mammary quarters presenting positive results to CMT and microbiological exams were considered as subclinical mastitis cases and these represented 20,03% among 734 studied samples. CMT negative results were the most frequent observed during the study (69,07%), independent of lactation stage or season of the year considered. Among these CMT negative milk samples, 57,58% were positive in microbiological exams. There was a high occurrence of contagious microorganisms among the 579 identified bacteria from 19 different genus, isolated in pure or in association. Corynebacterium spp. was the most prevalent (47,67%), in every lactation stage or season of the year. Considering the absence of a normal distribution, somatic cell counts were submitted to a logarithmic transformation, log2(CCS/100)+3, originating the transformed somatic cell count (TSCC). There were statistically significant differences between TCCS means from milk samples with positive (2,23) and negative (1,71) results to CMT. Such differences were also observed in milk... (Complete abstract click electronic access below) / Doutor

O uso de antibióticos para tratamento da mastite bovina subclínica durante a lactação apresenta restrições econômicas e a administração indiscriminada e inadequada destes medicamentos os torna potencialmente tóxicos aos animais e aos consumidores finais dos produtos lácteos. A utilização de medicamentos homeopáticos oferece menor custo, facilidade de administração, não há risco de resistência microbiana e não é necessário o descarte do leite dos animais em tratamento. Além disso, a homeopatia é reconhecida no Brasil como especialidade médica veterinária e aceita para uso no sistema de produção animal orgânico. Portanto, o presente trabalho teve como objetivo avaliar a eficácia de dois protocolos homeopáticos para tratamento da mastite bovina subclínica durante a lactação. O experimento foi realizado no período de seis meses e dividido em duas etapas. Na Etapa I, um grupo foi medicado com Sulphur 30CH, enquanto que o outro recebeu placebo. Na Etapa II, um grupo foi medicado com Calcarea carbonica 30CH e o outro grupo recebeu placebo. Os medicamentos foram administrados a cada trinta dias na Etapa I e a cada quinze dias na Etapa II. Em cada etapa foram coletadas amostras de leite antes, durante e após o tratamento. Para avaliar a eficácia dos protocolos, durante o experimento foram analisadas 138 amostras de leite na Etapa I e 72 amostras na Etapa II, realizando-se prova de Tamis, California Mastitis Test (CMT), contagem de células somáticas (CCS), quantificação de polimorfonucleares (PMN) e mononucleares (MN) por microscopia óptica, exame microbiológico e mensuração da produção leiteira. As análises estatísticas foram realizadas utilizando o software Graphpad Instat 1990-93. Os protocolos homeopáticos testados não diminuíram a celularidade do leite, pois não houve diferenças significativas nos resultados do CMT e da CCS no decorrer do experimento. Também não foi constatada nenhuma alteração significante na produção láctea. O microrganismo isolado com maior frequência no rebanho estudado foi o Corynebacterium bovis. Porém, não houve diferenças significativas em relação à frequência de isolamento deste microrganismo ao longo dos tratamentos, indicando a sua permanência nas glândulas mamárias. Além disso, observou-se durante todo o experimento o predomínio de células PMN em relação às MN. Sugere-se o estudo de novos protocolos homeopáticos com outros medicamentos, potência e frequência de administração, a fim de buscar alternativas para o tratamento de vacas com mastite subclínica / The use of antibiotics for treatment of bovine subclinical mastitis during lactation presents economic restrictions and the indiscriminate and inappropriate administration of these drugs makes them potentially toxic to animals and consumers of dairy products. The use of homeopathic medicine offers lower cost, ease of administration, no risk of microbial resistance and is not necessary to discard milk from animals under treatment. Furthermore, homeopathy is recognized as a medical specialty in Brazil and support for veterinary use in organic livestock production system. Therefore, this study aimed to evaluate the effectiveness of two protocols for homeopathic treatment of bovine subclinical mastitis during lactation. The experiment was performed between six months and divided into two stages. In Phase I, one group was treated with Sulphur 30CH, while the other received placebo. In Phase II, one group was treated with Calcarea carbonica 30CH and the other group received placebo. The drugs were administered every thirty days in the Phase I and every fifteen days in the Phase II. At each step the milk samples were collected before, during and after treatment. To evaluate the effectiveness of the protocols during the experiment were analyzed 138 samples of milk at Phase I and 72 samples at Phase II, performing proof Tamis, California Mastitis Test (CMT), somatic cell count (SCC), quantification of polymorphonuclear (PMN) and mononuclear (MN) by optical microscopy, microbiological examination and measurement of milk production. Statistical analysis was performed using the software Graphpad Instat 1990-93. The homeopathic protocols tested not diminished cellularity of milk because there were no significant differences in the results of CMT and SCC during the experiment. Nor was it found no significant change in milk production. The organism isolated most frequently in the herd studied was Corynebacterium bovis. However, no significant differences were observed in the frequency of isolation of this microorganism during the treatments, indicating his permanence in the mammary glands. Furthermore, it was observed during the experiment a predominance of PMN cells compared to MN. It is suggested the study of new protocols with other homeopathic medicines, potency and frequency of administration, to find alternatives for the treatment of cows with subclinical mastitis

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-26T14:20:58Z No. of bitstreams: 2 Dissertação - Ernani Flávio Lopes Barbosa - 2016.pdf: 2105902 bytes, checksum: 07f1df5e2ebb5676e0813d7755d23347 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-26T14:21:16Z (GMT) No. of bitstreams: 2 Dissertação - Ernani Flávio Lopes Barbosa - 2016.pdf: 2105902 bytes, checksum: 07f1df5e2ebb5676e0813d7755d23347 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-26T14:21:16Z (GMT). No. of bitstreams: 2 Dissertação - Ernani Flávio Lopes Barbosa - 2016.pdf: 2105902 bytes, checksum: 07f1df5e2ebb5676e0813d7755d23347 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-08-22 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Corynebacterium pseudotuberculosis is the etiologic agent of Caseous Lymphadenitis (CLA), a chronic infectious disease that is distributed worldwide and affects goats and sheep. The disease is characterized by the development of granulomes in superficial lymph nodes and some organs, as liver, spleen, lungs, and kidneys. The occurrence of CLA in Brazilian small ruminant herds is widely spread, causing economic losses due to a reduction in productive efficiency and in leather economic value, and occasional death of affected animals. The objective of this study was to identify the seroprevalence of specific anti-C. pseudotuberculosis antibodies in goats and sheep in the state of Goiás, and to correlate the infection with breeding procedures. For this, 1815 serum samples from goats at 213 production units and 751 sera samples from sheep at 113 rural properties were analyzed. The state of Goiás was divided in regional agencies, as proposed by the state government. These samples were submitted to an indirect ELISA technique for specific antibodies detection, and a questionnaire was applied to farmers in order to correlate the presence of specific antibodies with the breeding procedures adopted by these breeders. The data were analyzed using the R software. The results showed a CLA seroprevalence of 29.4% for sheep and 51.8% for goats. From the properties included in the study, 84 and 88.2% presented positive goats or sheep, respectively. Positive animals were present in 100% of the state regional agencies. 90% of the Goiás studied municipalities presented positive sheep, and CLA positive goats were present in 84.7% of the municipalities included in this survey. From the studied goats, 54.2% of the females showed positive results, and 44.7% of the males had the presence of C. pseudotuberculosis-specific antibodies. From the animalsat more than 36 months of age, the results showed prevalence of 62.5% and 62.8% of positive sheep and goats, respectively. When considering the type of workers at the production units, sheep farms where the owners employed the breeders presented 31.3% prevalence. However, when the owner’s family was the main breeders, the sheep presented 21.7% prevalence. 53.1% of the goats that were acquired from other farms presented positive results. For sheep herds, there was no significant correlation with breeding procedures. On the other hand, 54.4% of the goats that had signalgrass as the main forage were positive at the ELISA. Considering these results, it can be concluded that this study represents the first CLA epidemiological survey in goats and sheep from Goiás state, and the occurrence of the infection with the bacteria is widespread at the state. / A Linfadenite caseosa (LC) é uma doença infectocontagiosa de caráter crônico, causada pela bactéria Corynebacterium pseudotuberculosis. Este patógeno é de distribuição mundial, acomete principalmente caprinos e ovinos e a doença que caracteriza-se pela formação de granuloma em nódulos linfáticos superficiais, podendo acometer órgãos e linfonodos internos. No Brasil, a doença encontra-se presente em grande parte dos rebanhos de pequenos ruminantes, causando danos econômicos, pela perda de valor do couro, perda de eficiência produtiva e, ocasionalmente, morte dos animais infectados. O objetivo do estudo foi identificar a soroprevalência de anticorpos específicos para C. pseudotuberculosis em ovinos e caprinos do estado de Goiás e identificar fatores de risco relacionados à doença. Foram analisadas 1815 amostras de soro de ovinos oriundos de 212 propriedades e 751 amostras de caprinos de 113 propriedades. O estado de Goiás foi estratificado em 18 regionais, conforme estrutura organizacional da Agência Goiana de Defesa Agropecuária. As amostras de soro foram submetidas à técnica de ELISA indireto para a detecção de anticorpos e o questionário aplicado teve as variáveis analisadas quanto ao grau de significância do sistema produtivo, havendo relevância para sexo, faixa etária, tipo de mão de obra, procedência e pastagem. Os dados foram analisados estatisticamente pelo software R. Os resultados obtidos mostraram que 29,4% dos soros de ovinos eram sorologicamente positivos para a bactéria e 51,8% dos caprinos amostrados mostraram-se positivos. Do total de propriedades de ovinos e caprinos, 83,4% e 84,7% tiveram animais positivos em seus rebanhos, respectivamente. Pode-se afirmar que para ovinos, 90% dos 120 municípios tiveram rebanhos positivos e para caprinos, 84,7% dos 85 municípios amostrados. Em relação ao sexo, observou-se maior positividade para fêmeas (31,5%) do que para machos (23,5%), na espécie ovina. No que tange à espécie caprina, 54,2% das fêmeas foram positivas e 44,7% dos machos foram sororreagentes. Para a variável faixa etária com animais acima de 36 meses, observou-se que 62,5% dos ovinos e 62,8% de caprinos tiveram exposição ao patógeno. Analisando a variável tipo de mão de obra, pode-se afirmar que 31,3% dos ovinos manejados por funcionários contratados foram positivos. Por outro lado, ovinos cuja mão de obra correspondeu à familiar apresentaram menor percentual (21,7%). Quanto à procedência dos caprinos, 53,1% cuja origem foi externa mostraram-se positivos. Para o quesito pastagem destinada aos caprinos, observou-se que 54,4% que consumiram braquiária e outras espécies forrageiras explicitaram resultado positivo para o diagnóstico sorológico de linfadenite caseosa. Por tais resultados, pode-se afirmar que este estudo representa a primeira descrição soro epidemiológica para linfadenite caseosa em rebanhos ovinos e caprinos do estado de Goiás, sendo notória a disseminação de Corynebacterium pseudotuberculosis em todas as unidades regionais.

O uso de antibióticos para tratamento da mastite bovina subclínica durante a lactação apresenta restrições econômicas e a administração indiscriminada e inadequada destes medicamentos os torna potencialmente tóxicos aos animais e aos consumidores finais dos produtos lácteos. A utilização de medicamentos homeopáticos oferece menor custo, facilidade de administração, não há risco de resistência microbiana e não é necessário o descarte do leite dos animais em tratamento. Além disso, a homeopatia é reconhecida no Brasil como especialidade médica veterinária e aceita para uso no sistema de produção animal orgânico. Portanto, o presente trabalho teve como objetivo avaliar a eficácia de dois protocolos homeopáticos para tratamento da mastite bovina subclínica durante a lactação. O experimento foi realizado no período de seis meses e dividido em duas etapas. Na Etapa I, um grupo foi medicado com Sulphur 30CH, enquanto que o outro recebeu placebo. Na Etapa II, um grupo foi medicado com Calcarea carbonica 30CH e o outro grupo recebeu placebo. Os medicamentos foram administrados a cada trinta dias na Etapa I e a cada quinze dias na Etapa II. Em cada etapa foram coletadas amostras de leite antes, durante e após o tratamento. Para avaliar a eficácia dos protocolos, durante o experimento foram analisadas 138 amostras de leite na Etapa I e 72 amostras na Etapa II, realizando-se prova de Tamis, California Mastitis Test (CMT), contagem de células somáticas (CCS), quantificação de polimorfonucleares (PMN) e mononucleares (MN) por microscopia óptica, exame microbiológico e mensuração da produção leiteira. As análises estatísticas foram realizadas utilizando o software Graphpad Instat 1990-93. Os protocolos homeopáticos testados não diminuíram a celularidade do leite, pois não houve diferenças significativas nos resultados do CMT e da CCS no decorrer do experimento. Também não foi constatada nenhuma alteração significante na produção láctea. O microrganismo isolado com maior frequência no rebanho estudado foi o Corynebacterium bovis. Porém, não houve diferenças significativas em relação à frequência de isolamento deste microrganismo ao longo dos tratamentos, indicando a sua permanência nas glândulas mamárias. Além disso, observou-se durante todo o experimento o predomínio de células PMN em relação às MN. Sugere-se o estudo de novos protocolos homeopáticos com outros medicamentos, potência e frequência de administração, a fim de buscar alternativas para o tratamento de vacas com mastite subclínica / The use of antibiotics for treatment of bovine subclinical mastitis during lactation presents economic restrictions and the indiscriminate and inappropriate administration of these drugs makes them potentially toxic to animals and consumers of dairy products. The use of homeopathic medicine offers lower cost, ease of administration, no risk of microbial resistance and is not necessary to discard milk from animals under treatment. Furthermore, homeopathy is recognized as a medical specialty in Brazil and support for veterinary use in organic livestock production system. Therefore, this study aimed to evaluate the effectiveness of two protocols for homeopathic treatment of bovine subclinical mastitis during lactation. The experiment was performed between six months and divided into two stages. In Phase I, one group was treated with Sulphur 30CH, while the other received placebo. In Phase II, one group was treated with Calcarea carbonica 30CH and the other group received placebo. The drugs were administered every thirty days in the Phase I and every fifteen days in the Phase II. At each step the milk samples were collected before, during and after treatment. To evaluate the effectiveness of the protocols during the experiment were analyzed 138 samples of milk at Phase I and 72 samples at Phase II, performing proof Tamis, California Mastitis Test (CMT), somatic cell count (SCC), quantification of polymorphonuclear (PMN) and mononuclear (MN) by optical microscopy, microbiological examination and measurement of milk production. Statistical analysis was performed using the software Graphpad Instat 1990-93. The homeopathic protocols tested not diminished cellularity of milk because there were no significant differences in the results of CMT and SCC during the experiment. Nor was it found no significant change in milk production. The organism isolated most frequently in the herd studied was Corynebacterium bovis. However, no significant differences were observed in the frequency of isolation of this microorganism during the treatments, indicating his permanence in the mammary glands. Furthermore, it was observed during the experiment a predominance of PMN cells compared to MN. It is suggested the study of new protocols with other homeopathic medicines, potency and frequency of administration, to find alternatives for the treatment of cows with subclinical mastitis

Corynebacterium poinsettiae mutant strains blocked in carotenoid biosynthesis were obtained by treatment with the mutagen N-methyl-N1-nitro-N-nitrosoguanidine. Additional carotenoid (Crt) mutant strains were obtained from a previous study conducted in our laboratory. Fifty-nine Crt mutants affected in carotenoid biosynthesis were examined by a normal phase high performance liquid chromatography (HPLC) system. Pigment extracts of Crt mutants and C. poinsettiae wild type strains were resolved by an isocratic system with hexane:acetone:dicholoromethane, 11.35:1.73:1.00 (by vol.) as the eluting solvent. In addition to the five major peaks, twelve minor peaks were observed in the wild type C. poinsettiae strain used in this study. Crt mutant and wild type strain peak heights were measured from the individual chromatograms and the peak height data set created was analyzed using the Statistical Analysis System program to perform a cluster analysis. The cluster analysis revealed five carotenoid mutant groups. Carotenoid pigments which accumulated or were absent in each of the cluster groups are reported. Cluster group 1 mutants (CrtA) are blocked in the dehydrogenase(s) which is(are) responsible for the dehydrogenations between phytoene and lycopene. Cluster group 2 mutants (CrtB) appear to be blocked at a second dehydrogenase specific for the dehydrogenation from C.p. 470 to C.p. 496. Cluster group 3 mutants (CrtC) are blocked at a cyclization step in the pathway which involves cyclization of C.p. 496 to C.p. 470 and which may cyclize C.p. 473 to C.p. 450. The genes CrtA and CrtB map only 0.5 map units from each other while CrtA and CrtC map 2.1 map units from one another. Mutants which accumulate end products but which lack certain precursors indicate a branched pathway for pigment biosynthesis exists in this organism. Media for the formation, fusion and regeneration of C. poinsettiae protoplasts are reported and a protocol for the use of these media in genetic crosses of strains blocked in carotenoid biosynthesis is described. While isolating antibiotic resistant mutants useful in genetic analyses, novobiocin resistant mutants were observed to have a distinctly different colony pigment phenotype as compared to the wild type strain. HPLC chromatograms of a novobiocin resistant strain showed a distinctly different carotenoid pigment profile. The results provide evidence for differential gene expression in the carotenoid biosynthetic pathway when these mutants are grown in the presence of novobiocin.

The purpose of this study was to completely characterize the protein moiety in the caroteno complex in C. poinsettae, determine if the distribution and level of protein in the pigment-protein complex in membranes of the wild type and in a colorless mutant could account for the differences in the stability of the membrane, and to determine if this protein is common to other pigmented and non-pigmented organisms. Also, electron microscopy of cell membranes of C. poinsettiae which had been exposed to gold-labelled antibody against the protein moitey of the pigment-protein complex, demonstrating that the protein is randomly distributed in the membranes of both wild type and colorless mutant.

The purpose of this study was (1) to determine whether a correlation exists among the protein profiles, extracted from cell membranes of mutants belonging to five pigment cluster groups, (2) to locate the protein moiety and cartenoprotein complex in the membranes of wild type and colorless mutant (designated W-19) of C. poinsettae and to show whether there are any structural differences between cell membranes of the wild type and a colorless mutant, (3) to determine the effect of six antibiotics on cartenoid gene expression.

In a minimal medium supplemented with glucose and yeast extract, the optimum pH for the growth of C. poinsettias was found to be 7.5. The organism requires thiamine, biotin, and pantothenic acid for growth. No absolute requirement was found for any amino acid, purine or pyrimidine although an amino acid mixture was stimulatory. Casamino acids could be substituted for the synthetic amino acid mixture. Yeast extract provided an additional factor(s) necessary for maximal growth. The results suggest that the unknown factor found in yeast extract might be purified by a combination of solvent extraction, and adsorption and elution from charcoal.

Carotenoid pigments were extracted from Corynebacteriuma poinsettiae (wild type) ATCC 9682, and from 108 mutants obtained by exposure of a streptomycin resistant strain of C. poinsettiae to ultra-violet light irradiation and N-methyl- N'-nitro-N-nitrosoguanidine. The pigments were characterized by their absorption maxima, Rf-values, and partition ratios in petroleum ether and methanol. Thin layer chromatography was used to compare pigments of the wild type with those of the mutants. Possible biosynthetic pathways in carotenoid synthesis of the wild type were postulated on the basis of the observed genetic blocks. Mutants were found which suggested the existence of a linear pathway in carotenoid synthesis from the aliphatic C4 0 molecule to the bi-cyclic C50-diol. Other mutants suggested possible alternative pathways in the biosynthesis of these pigments or the presence of intermediates not detectable by thin layer chromatography.

This investigation studies effects of environmental factors on growth and carotenogenesis in Corynebacterium strain 7ElC. Changes in pH were found to effect growth more than carotenogenesis. However, certain nutrients or long incubation periods stimulated carotenoid formation more than growth. Dark conditions in a mineral salts-glucose medium stimulated growth, but minimized carotenogenesis. Tryptic soy broth or yeast extract elicited carotenogenesis in darkness. Although brief light exposure during inoculation was photoinductive, continuous exposure to light following inoculation was required for maximum pigment synthesis. Dark grown stationary phase cells required 24-hours of light for maximum pigment synthesis. Chloramphenicol inhibition of carotenogenesis in dark grown cells exposed to light showed that enzymes needed for carotenoid synthesis were absent from dark grown cultures.