En Amérique du Nord, et particulièrement au Canada, il y a très peu de données sur l’incidence des causes de mortalité chez l’espèce caprine. Le premier objectif de cette étude était de déterminer les principales causes de mortalité chez les chèvres au Québec. Depuis 2006, avec l’arrêt de la vaccination contre la lymphadénite caséeuse, les éleveurs de caprins laitiers et de boucherie du Québec ont rapporté une recrudescence des abcès chez leur bétail. Le second but de cette étude était de déterminer l’importance de la lymphadénite caséeuse dans le dépérissement et la mortalité des chèvres du Québec. Cent-cinquante-deux chèvres provenant de 13 élevages différents ont été soumises pour nécropsie et la cause de mortalité, de même que la présence d’abcès (s’il y a lieu), leur localisation et leur cause furent compilés. Les mortalités proportionnelles étaient, par ordre décroissant : l’entérotoxémie de type D (n= 26; 17,1%), la pneumonie (n= 21; 13,8%), la paratuberculose (n= 16; 10,5%), listériose encéphalitique (n= 10; 6,6%), la toxémie de gestation (n= 8; 5,3%), l’arthrite-encéphalite caprine (n= 7; 4,6%) et la lymphadénite caséeuse (n= 6; 3,9%). La lymphadénite caséeuse a été diagnostiquée chez 24,3% des chèvres soumises, mais sans être une cause majeure de dépérissement ou de mortalité. Les abcès étaient internes dans 54,1% des cas. Au total, la paratuberculose a été diagnostiquée chez 29 chèvres (16 en étant décédées) et fut considérée comme une cause majeure de dépérissement, d’émaciation et de mortalité. Le développement et l’implantation de mesures préventives contre cette maladie seraient donc à envisager dans le futur. / In North America, and especially in Canada, the incidence and the causes of death in goats are not well documented. The first objective of this study was to determine the main causes of mortality in goats in Quebec. Dairy and meat goat farmers had reported an increase in abscesses in their herd since vaccination for caseous lymphadenitis stopped being available (2006) in Quebec. The second objective of this study was to determine the importance of caseous lymphadenits in wasting and mortality in caprine herds from Quebec. One hundred and fifty-two goats from 13 different herds from Quebec were submitted for necropsy and the cause of mortality, as well as the presence, location and cause of abscesses (if present) were recorded. Proportional mortalities were, in decreasing order: type D enterotoxemia (n= 26; 17,1%), pneumonia (n= 21; 13,8%), paratuberculosis (n= 16; 10,5%), encephalitic listeriosis (n= 10; 6,6%), pregnancy toxemia (n= 8; 5,3%), caprine arthritis-encephalitis (n= 7; 4,6%) and caseous lymphadenitis (n= 6; 3,9%). Caseous lymphadenitis was diagnosed in 24.3% of the submitted goats, but was not a major cause of debilitation or mortality. Abscesses were localized internally in 54.1% of the cases. Overall, paratuberculosis was diagnosed in 29 goats (16 of which died of the condition) and was considered a major cause of wasting, emaciation and mortality. Development and implementation of preventive measures to control this disease in goat herds would certainly be relevant in the future.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado. Por outro lado, o BCGr pUS977dtbPW8, quando submetido a seis sub-cultivos consecutivos in vitro não apresentou diferença significativa na capacidade de expressar o DTB, demonstrando portanto a persistência da estabilidade funcional da linhagem recombinante. A estabilidade estrutural da construção pUS977dtbPW8 também foi avaliada por PCR confirmando a presença do gene dtb em colônias do BCGr pUS977dtbPW8 . Adicionalmente, foi possível avaliar preliminarmente in vitro a capacidade soroneutralizante dos soros de camundongos imunizados com BCGr pUS977dtbPW8 após 30 e 60 dias em células VERO. A ação citotóxica da toxina diftérica entre as diluições de 1/4 e 1/16 foram neutralizadas com o pool de soros imunes com 60 dias. Finalmente, em nosso estudo foi possível avaliar o potencial da vacina BCG como vetor de expressão de um antígeno de Corynebacterium diphtheriae in vitro e in vivo. / The diphtheria vaccine currently used in Brazil (DTP), despite its history of high efficacy in the prevention of diphtheria, is associated with episodes of toxicity and vaccine reactogenicity in the vaccinee, resulting from the presence in the vaccine of residual proteins derived from the production process or detoxification. Strategies for the development of new vaccines more effective and economically viable against diphtheria have been the subject of intense investigation. The alternative proposed by our group is the use of the vaccine against tuberculosis (Mycobacterium bovis BCG Moreau sub strain) as a vector for the gene that encodes the 58.3 kDa fragment B of the diphtheria toxin (DTB). In our project the dtb gene was cloned into the bifunctional vector pUS977 for cytoplasmic expression and recombinant BCG (rBCG) clones, selected after transformation of BCG, were tested for expression of the DTB polypeptide and antigenicity against polyclonal antibodies anti- diphtheria toxoid by immunoblotting. The integrity and identity of the DNA sequence encoding the dtb gene carried by the plasmid construct pUS977dtbPW8 was confirmed by DNA Sequencing and Analysis of Similarity. The immunogenicity of the rBCG expressing the DTB was investigated in BALB/c mice and the results revealed a specific seroconversion (IgG). Also, infectivity and microbicidal activity were analyzed in the intracellular environment by infecting human monocytes (THP-1 cell line) with rBCG. The data obtained indicated intracellular survival within 12 days. In this context, splenocytes collected from mice at days 30 and 60 after immunization were removed and assayed for live bacteria. The results showed that rBCG persisted viable up to 60 days in the absence of selective pressure and cell viable counts did not change significantly during testing. Additionally, the rBCG subjected to six consecutive sub-cultures in vitro showed no significant difference in the ability to express the DTB, thus demonstrating the functional stability of the recombinant vaccine. The structural stability of the construct pUS977dtbPW8 was also confirmed by PCR detection of the dtb gene in rBCG colonies. Also, it was possible to have a preliminary evaluation of the neutralizing capacity of sera from mice immunized with BCGr 30 and 60 days after immunization. The cytotoxic action of diphtheria toxin, between dilutions 1/ 4 and 1/16, was neutralized by mice sera in an in vitro assay using VERO cells. Finally, in our study it was possible to evaluate the potential of BCG as a vector for expression of an antigen of Corynebacterium diphtheriae in vitro and in vivo.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado. Por outro lado, o BCGr pUS977dtbPW8, quando submetido a seis sub-cultivos consecutivos in vitro não apresentou diferença significativa na capacidade de expressar o DTB, demonstrando portanto a persistência da estabilidade funcional da linhagem recombinante. A estabilidade estrutural da construção pUS977dtbPW8 também foi avaliada por PCR confirmando a presença do gene dtb em colônias do BCGr pUS977dtbPW8 . Adicionalmente, foi possível avaliar preliminarmente in vitro a capacidade soroneutralizante dos soros de camundongos imunizados com BCGr pUS977dtbPW8 após 30 e 60 dias em células VERO. A ação citotóxica da toxina diftérica entre as diluições de 1/4 e 1/16 foram neutralizadas com o pool de soros imunes com 60 dias. Finalmente, em nosso estudo foi possível avaliar o potencial da vacina BCG como vetor de expressão de um antígeno de Corynebacterium diphtheriae in vitro e in vivo. / The diphtheria vaccine currently used in Brazil (DTP), despite its history of high efficacy in the prevention of diphtheria, is associated with episodes of toxicity and vaccine reactogenicity in the vaccinee, resulting from the presence in the vaccine of residual proteins derived from the production process or detoxification. Strategies for the development of new vaccines more effective and economically viable against diphtheria have been the subject of intense investigation. The alternative proposed by our group is the use of the vaccine against tuberculosis (Mycobacterium bovis BCG Moreau sub strain) as a vector for the gene that encodes the 58.3 kDa fragment B of the diphtheria toxin (DTB). In our project the dtb gene was cloned into the bifunctional vector pUS977 for cytoplasmic expression and recombinant BCG (rBCG) clones, selected after transformation of BCG, were tested for expression of the DTB polypeptide and antigenicity against polyclonal antibodies anti- diphtheria toxoid by immunoblotting. The integrity and identity of the DNA sequence encoding the dtb gene carried by the plasmid construct pUS977dtbPW8 was confirmed by DNA Sequencing and Analysis of Similarity. The immunogenicity of the rBCG expressing the DTB was investigated in BALB/c mice and the results revealed a specific seroconversion (IgG). Also, infectivity and microbicidal activity were analyzed in the intracellular environment by infecting human monocytes (THP-1 cell line) with rBCG. The data obtained indicated intracellular survival within 12 days. In this context, splenocytes collected from mice at days 30 and 60 after immunization were removed and assayed for live bacteria. The results showed that rBCG persisted viable up to 60 days in the absence of selective pressure and cell viable counts did not change significantly during testing. Additionally, the rBCG subjected to six consecutive sub-cultures in vitro showed no significant difference in the ability to express the DTB, thus demonstrating the functional stability of the recombinant vaccine. The structural stability of the construct pUS977dtbPW8 was also confirmed by PCR detection of the dtb gene in rBCG colonies. Also, it was possible to have a preliminary evaluation of the neutralizing capacity of sera from mice immunized with BCGr 30 and 60 days after immunization. The cytotoxic action of diphtheria toxin, between dilutions 1/ 4 and 1/16, was neutralized by mice sera in an in vitro assay using VERO cells. Finally, in our study it was possible to evaluate the potential of BCG as a vector for expression of an antigen of Corynebacterium diphtheriae in vitro and in vivo.

Abstract Regulation of transcription by extracytoplasmic-function (ECF) sigma factors of RNA polymerase is an efficient way of cell adaptation to diverse environmental stresses. Amino acid-producing gram-positive bacterium Corynebacterium glutamicum codes for seven sigma factors: the primary sigma factor SigA, the primary-like sigma factor SigB and five ECF stress- responsive sigma factors (SigC, SigD, SigE, SigH and SigM). The sigH gene encoding SigH sigma factor is located in a gene cluster together with the rshA gene, encoding the anti-sigma factor of SigH. Anti-sigma factors bind to their cognate sigma factors and inhibit their transcriptional activity. Under the stress conditions the binding is released allowing the sigma factors to bind to the RNAP core enzyme. In this thesis, regulation of expression of genes encoding the most important ECF sigma factor SigH and its anti-sigma factor RshA as well as genes belonging to the SigH-regulon were mainly studied. The transcriptional analysis of the sigH-rshA operon revealed four housekeeping promoters of the sigH gene and one SigH-dependent promoter of the rshA gene. For testing the role of the complex SigH-RshA in gene expression, the C. glutamicum ΔrshA strain was used for genome-wide transcription profiling with DNA Microarrays technique under...

Submitted by LIEGE ABDALLAH KAWAI null (liegekawai@gmail.com) on 2017-12-13T13:56:50Z No. of bitstreams: 1 liege kawai - tese final.pdf: 5173025 bytes, checksum: 74d38826e72cc45ba04c0050ac6a409b (MD5) / Approved for entry into archive by Elza Mitiko Sato null (elzasato@ibilce.unesp.br) on 2017-12-13T14:57:31Z (GMT) No. of bitstreams: 1 kawai_la_dr_sjrp.pdf: 5173025 bytes, checksum: 74d38826e72cc45ba04c0050ac6a409b (MD5) / Made available in DSpace on 2017-12-13T14:57:31Z (GMT). No. of bitstreams: 1 kawai_la_dr_sjrp.pdf: 5173025 bytes, checksum: 74d38826e72cc45ba04c0050ac6a409b (MD5) Previous issue date: 2017-11-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Corynebacterium pseudotuberculosis (C. pseudotuberculosis), é uma bactéria gram positiva anaeróbia facultativa, pleomórfica, que não esporula, não forma cápsula, e que apresenta 2 biotipos ou biovares, sendo o biovar equi capaz de infectar preferencialmente equinos, enquanto o biótipo denominado ovis acomete pequenos ruminantes. Esta bactéria faz parte do grupo CMNR (Corynebacterium, Mycobacterium, Nocardia e Rhodococcus), que demonstra grande importância veterinária e médica, uma vez que estes micro-organismos comumente infectam animais, podendo infectar o homem, causando perdas econômicas pela ineficácia ou alto custo de terapias existentes. Um exemplo seria a linfadenite caseosa (LC) causada por C. pseudotuberculosis, que afeta particularmente a pecuária de caprinos e ovinos, com a condenação da carcaça e redução da produção de lã e de carne. A transmissão da doença e contágio dos animais é direta, muitas vezes através da alimentação e ingestão de água em local contaminado por animais doentes. Essa doença possui incidência na pecuária mundial, principalmente de caprinos e ovinos, havendo registros de ocorrência no Brasil, Europa, Oriente Médio, Austrália, Nova Zelândia, África do Sul, Canadá e Estados Unidos e mesmo com todos os avanços tecnológicos, ainda não há métodos de prevenção totalmente eficazes, como vacinas e medicamentos, tampouco para o tratamento de animais infectados, que geralmente são de custo elevado, por longos períodos e sem a garantia de cura ou de isenção de reincidência da LC. Deste modo, técnicas mais rápidas e fáceis para detecção e diagnóstico da doença, bem como para seu tratamento, se tornam imprescindíveis, evitando não só a disseminação da doença, mas também suas consequentes perdas econômicas. Atualmente, devido ao uso indiscriminado de antibióticos para o tratamento de infecções de origem bacteriana, bem como à constante exposição destes micro-organismos a essas drogas em ambientes hospitalares, observa-se o desenvolvimento de micro-organismos resistentes às terapias disponíveis, sendo um desafio mundial a descoberta e elaboração de tratamentos eficazes para a prevenção, controle e eliminação destes patógenos, como alternativa aos já existentes. Visando terapias alternativas e direcionadas para infecção por C. pseudotuberculosis, as proteínas tioredoxina, tioredoxina redutase e diphtheria toxin repressor foram estudadas no presente trabalho, a fim de melhor compreender este micro-organismo. / Corynebacterium pseudotuberculosis (C. pseudotuberculosis), is a gram-positive, facultative anaerobe, pleomorphic, non-sporulating bacterium with two biotopes or biovars, being the biovar equi capable of infecting horses, whereas the biotype called ovis infects small ruminants. It is part of the CMNR group (Corynebacterium, Mycobacterium, Nocardia and Rhodococcus), which demonstrates great veterinary and medical importance, since these common microorganisms infect animals and can infect humans, causing economic losses due to the inefficiency or high cost of existing therapies. An example is a caseous lymphadenitis (LC) caused by C. pseudotuberculosis, which particularly affects the goats and sheep livestock, with carcass condemnation and reduction of wool and meat production. The transmission of disease and the contagion of animals is direct, often through feeding and drinking water in places contaminated by sick animals. This disease has an incidence in the world livestock, mainly of goats and sheep, with occurrence records in Brazil, Europe, the Middle East, Australia, New Zealand, South Africa, Canada and the United States and even with all technological advances, still there are no totally effective prevention methods, such as vaccines and medications, nor for the treatment of infected animals, which are usually of a high cost, for long periods and without guarantee of cure or exemption from recurrence of LC. In this way, faster and easier techniques for the detection of the diagnosis of this disease as well as for its treatment become essential, avoiding not only a spread of the disease, but also its consequent economic losses. Currently, the use of indiscriminate antibiotics for the treatment of infections of bacterial origin, as well as the constant exposure of these microorganisms to these drugs in hospital environments, shows the development of microorganisms resistant to the available therapies, being one world-wide challenge the elaboration of effective treatments for the prevention, control and elimination of these pathogens, as an alternative to the existing ones. Aiming alternative therapies for infection by C. pseudotuberculosis, proteins such as thioredoxin, thioredoxin reductase, diphtheria toxin repressor, were studied in the present work, for a better comprehension of this microorganism.

Submitted by (edna.saturno@ufrpe.br) on 2016-10-07T14:44:50Z No. of bitstreams: 1 Flavia Figueiraujo Jabour.pdf: 2776762 bytes, checksum: c6f5ab38588135603c3ae6a6a04e7a81 (MD5) / Made available in DSpace on 2016-10-07T14:44:50Z (GMT). No. of bitstreams: 1 Flavia Figueiraujo Jabour.pdf: 2776762 bytes, checksum: c6f5ab38588135603c3ae6a6a04e7a81 (MD5) Previous issue date: 2013-02-28 / Tuberculosis, paratuberculosis and caseous lymphadenitis are important granulomatous diseases that affect various animal species, including sheep and goats and determine major economic losses. These diseases have similar clinical and pathological characteristics and deserve further investigation and differentiated to facilitate its control. The objective was to study to diagnose the occurrence of granulomatous diseases such as tuberculosis, paratuberculosis and caseous lymphadenitis in sheep and goats slaughtered in a municipal slaughterhouse, State of Alagoas, Brazil. Two hundred animals had been slaughtered during four visits made to the slaughterhouse from 2009 to 2011. Among these two hundred animals, 39 sheep and 3 goats presented macroscopic lesions in several body tissues. Histopathological, immunohistochemistry and bacteriological exams were executed for diagnostic. Lesions in the gut compatible with alterations caused by Oesophagostomum sp. were found in the sheep and goats. All samples were negative in immunohistochemistry for Mycobacterium spp. In the bacteriological test, Corybacterium pseudotuberculosis was isolated from lung, lymph nodes, small and large gut of 18 sheep and from the large gut of one goat. Alterations of tuberculosis and paratuberculosis were not observed, however lesions of other economically important diseases were found, such as caseous lymphadenitis associated with alterations of oesophagostomiasis e fascioliasis. In the second studythe objective was to perform the tuberculin skin test in 275 sheep and 45 goats coming from four herds in three cities of Eastern Mesoregion of Alagoas state. We also applied a questionnaire to analyze the epidemiological profile of the properties studied. All goats were negative to the tuberculin skin test and two sheep (0.72%) were positive for two different properties (50%). Join the occurrence of tuberculosis in sheep in the state of Alagoas. Despite the lower frequency of cases there were a high number of outbreaks. This result indicates that one should intensify their efforts to control tuberculosis in these regions studied to prevent the spread of mycobacteria to other flocks of sheep and goats and similar epidemiological studies should be conducted in other regions of the state of Alagoas to investigate the occurrence of disease and your impact in sheep and goat herds. / Tuberculose, paratuberculose e linfadenite caseosa são doenças granulomatosas importantes que acometem várias espécies animais, incluindo ovinos e caprinos e determinam grandes prejuízos econômicos. Estas doenças possuem características clínico-patológicas semelhantes e merecem ser melhor investigadas e diferenciadas para facilitar seu controle. Objetivou-se estudar a ocorrência de doenças granulomatosas em ovinos e caprinos abatidos em um matadouro no Estado de Alagoas, além de determinar a ocorrência de tuberculose em ovinos e caprinos em propriedades rurais na região da Zona da Mata Alagoana. No primeiro estudo Foram estudados 200 animais abatidos nos anos de 2009 a 2011. Destes, 39 ovinos e três caprinos apresentaram lesões macroscópicas em vários tecidos. Para o diagnóstico foram realizados exames histopatológico e bacteriológico, e imunohistoquímica. Observou-se, ao exame histopatológico, lesões intestinais compatíveis com Oesophagostomum sp. em ovinos e caprinos e com fasciolose no fígado de ovinos. Na imunohistoquímica todas as amostras foram negativas para Mycobacterium spp. Ao exame bacteriológico isolou-se Corynebacterium pseudotuberculosis nos tecidos de 18 ovinos (pulmão, fígado, linfonodos, intestino delgado e intestino grosso) e de um caprino (intestino grosso). Não foi diagnosticada tuberculose e paratuberculose nos animais estudados, no entanto observou-se a ocorrência de outras doenças economicamente importantes como linfadenite caseosa associada a lesões de oesofagostomose e fasciolose. No segundo estudo objetivou-se realizar o diagnóstico da tuberculose em caprinos e ovinos em três municípios da Mesorregião do Leste Alagoano. Foram tuberculinizados 275 ovinos e 45 caprinos procedentes de quatro rebanhos. Também foram aplicados questionários para estudar o perfil epidemiológico das propriedades estudadas. Todos os caprinos foram negativos ao teste tuberculínico e dois ovinos (0,72%) foram positivos, registrando-se 50% de focos da doença. Registra-se a ocorrência da tuberculose em ovinos no estado de Alagoas. Apesar da baixa frequência de casos positivos observou-se um elevado número de focos da doença. Este resultado indica que medidas de controle devem ser intensificadas para evitar a disseminação de micobactérias a outros rebanhos de ovinos e caprinos e estudos epidemiológicos semelhantes devem ser realizados em outras regiões do estado de Alagoas para investigar a ocorrência da doença e seu impacto na criação de caprinos e ovinos. Apesar de não ter sido diagnosticada a tuberculose clínica e a paratuberculose nos animais estudados, observou-se a ocorrência de outras doenças economicamente importantes como linfadenite caseosa associada a lesões de oesofagostomose e fasciolose nos animais abatidos.

Caseous lymphadenitis (CLA) is a disease primarily in sheep and goats, caused by Corynebacterium pseudotuberculosis, characterized by abscess formation in external and/or internal lymph nodes and organs. Economic losses occur due to emaciation, reduced milk production and impaired growth in affected animals. At the Swedish Veterinary Agency, bacterial culture is used as a diagnostic method for detecting CLA, requiring puncture of clinical abscesses. This sampling method increases the risk for disease transmission and overlooks animals affected by internal abscesses. In this study, a commercially available enzyme-linked immunosorbent assay (ELISA)-kit, based on an indirect ELISA, was validated for detecting antibodies for CLA, aiming for a more cost-effective and safer method that also can detect animals with internal abscesses. Serum and milk samples were analyzed to investigate the concordance between the two sample types. A subset of serum and milk samples was sent to Norway for analysis to compare the ELISA results, and comparison with another laboratory's results was conducted. The ELISA-kit demonstrated high sensitivity (92% for serum, 100% for milk), crucial for controlling CLA at herd level, while the specificity was estimated at 68% for serum and 61% for milk. To avoid increased and unnecessary culling of animals, a confirmatory method should analyze positive individuals to increase the specificity. Pooled milk samples could reduce costs for herd-level analysis, followed by serum sampling for positive herds.

Lors de ce travail, une étude comparative entre trois souches de C. glutamicum a été réalisée. Celles-ci sont C. glutamicum 2262, une souche surproductrice de glutamate suite à l’élévation de température du milieu de culture de 33 à 39°C, C. glutamicum 2262 NP un variant incapable d’excréter du glutamate dans ces mêmes conditions et C. glutamicum 2262 ∆pks13 un mutant dépourvu de bicouche mycolique externe. Un modèle métabolique original reprenant les différentes modifications physiologiques aboutissant à l’excrétion du glutamate au cours du procédé thermo-induit a été établi. La bicouche mycolique joue un rôle primordial puisque son absence affecte sévèrement la production du glutamate. Dans un premier temps, l’élévation de température serait ressentie au niveau de cette bicouche. Ce ressenti, visualisé par l’accumulation de protéines caractéristiques d’un stress thermique, est nécessaire pour que la bactérie soit en capacité de surproduire le glutamate. Par la suite, la production de glutamate est régulée au niveau de l’α-cétoglutarate déshydrogénase (ODH) grâce à la phosphoprotéine OdhI. Suite au changement de température, celle-ci est déphosphorylée ce qui lui permet d’interagir avec ODH et de provoquer l’inhibition de cette dernière. Ceci se traduit par la redirection sur flux carboné vers la synthèse du glutamate. Aucun de ces évènements n’est observé chez C. glutamicum 2262 ∆pks13. Par ailleurs, l’élévation de température induit une modification de la composition de l’enveloppe cellulaire qui semble intervenir dans le processus physiologique aboutissant à l’excrétion du glutamate puisque très peu de changements sont observés chez C. glutamicum 2262 NP / During this work, a comparative study between three strains of Corynebacterium glutamicum was carried out. These strains were C. glutamicum 2262 which overproduces glutamate after an increase in the culture temperature from 33 to 39°C, C. glutamicum 2262 NP which is unable to produce glutamate in the same culture conditions and C. glutamicum 2262 ∆pks13 devoid of outer corynomycolic acid bilayer. An original metabolic model describing the successive physiological modifications responsible for the glutamate excretion during the temperature-triggered process was established. The presence of the corynomycolic acid bilayer appeared to be necessary since its lack affected dramatically the glutamate production. The temperature increase would be first sensed at the level of the external corynomycolic acid layer. This sensing was visualised through the accumulation of thermal stress proteins. In C. glutamicum 2262 ∆pks13, the synthesis of these proteins was not induced. The glutamate production is regulated at the oxoglutarate dehydrogenase (ODH) level by the phosphoprotein OdhI. A consequence of the temperature increase was the dephosphorylation of this regulatory protein and its interaction with ODH, provoking its inhibition. The carbon flux was then reoriented toward the glutamate synthesis. In C. glutamicum 2262 ∆pks13, no dephosphorylation of OdhI and no change in the ODH activity were not determined. The thermal stress also induced a change in the composition of the corynomycolic acid layer which was correlated with the ability of C. glutamicum 2262 to overproduce glutamate. In C. glutamicum 2262 NP, the composition of the corynomycolic acid layer remained unchanged

The effects of light, light "mimicking" chemicals, and protein synthesis inhibitors on the photo-induced carotenogenesis of Corynebacterium 7EIC were studied. Changes in the dosage of fluorescent light applied to dark grown cells showed a dose related carotenogenic response. Maintaining the same dosage but varying the wavelength of monochromatic light revealed that light with a wavelength of 280 to 450nm was responsible for photo-induction. It further showed a peak of photo-induction between the wavelengths of 370 and 430nm. The light "mimicking" chemicals antimycin A and p-Chloromercurybenzoate were shown to have no light "mimicking" effects. The transcriptional inhibitor of protein synthesis actinomycin D partially inhibited, and chloramphenicol a translational inhibitor, completely inhibited photo-induced carotenogenesis.

The aim of the thesis was to characterize chosen expression vectors used in biotechnologically important bacterial species, Corynebacterium glutamicum, and to test their use in studies of promoter activity control by sigma factors of RNA polymerase. Different properties of these vectors (level of expression of the cloned gene, leaky expression without inducer, dependence of expression level on inducer concentration and cell population homogeneity) were found by determination of expression level of the model gfpuv gene by fluorescence intensity assay of the produced protein and by gfpuv-expressing C. glutamicum cell population analysis using flow cytometry. The vector pEC-XT99A was chosen for testing the bi-plasmid system for assignment of a sigma factor to the chosen promoter. Although the level of expression provided by pEC-XT99A was not high, the vector showed no leaky expression, expression from the vector was comparable for a wide range of IPTG concentrations and the cell population was homogenous concerning the gene expression. Using pEC-XT99A from which individual stress sig genes were expressed, the σD factor was clearly assigned to the up-to-now unknown Pcg0420 promoter. Another vector for isolation and purification of C. glutamicum proteins was used to express the C. glutamicum sigM gene and to...