Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

A Diabetes mellitus do tipo I (IDDM), doença metabólica caracterizada pela deficiência na produção de insulina, causa profundas alterações sistêmicas, incluindo a cavidade bucal. A doença periodontal, que se apresenta mais agressiva em pacientes diabéticos, até mesmo na infância, tem como agentes etiológicos microrganismos denominados periodontopatógenos, dentre eles o Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), anteriormente chamado Actinobacillus actinomycetemcomitans, que em associação às alterações na resposta imune e inflamatória decorrentes da diabetes, poderá causar maior reabsorção do osso alveolar precocemente em crianças. A identificação precoce destes agressores poderá ser extremamente útil para a instituição de medidas preventivas e terapêuticas que possam inibir ou minimizar os efeitos nocivos da IDDM e suas conseqüências para a cavidade bucal. Para este fim, métodos moleculares de investigação estão sendo cada vez mais utilizados, destacando-se a Reação em Cadeia da Polimerase em Tempo Real (Real-Time PCR) com alta especificidade e sensibilidade para detectar e quantificar microrganismos, mesmo em pequeno número e em meios, até então, pouco explorados como a saliva. Por fim, a utilização de um antimicrobiano seguro, que possa ser aplicado topicamente por meio da escovação dentária, como o metronidazol 25% na forma de gel, poderia reduzir ou eliminar o A. actinomycetemcomitans evitando a progressão da doença periodontal. Assim, este estudo foi realizado para observar a presença do A. actinomycetemcomitans antes e após o uso do gel de metronidazol 25% uma vez ao dia, em substituição a uma das escovações diárias. Avaliou também a influência deste protocolo de tratamento nos índices de placa, gengival e profundidade de sondagem em 32 crianças, na faixa etária de 3 a 12 anos de idade, divididas em dois grupos: diabéticos tipo I (D) e não diabéticos (ND). Para avaliação da presença do A. actinomycetemcomitans foi utilizado o método da Real-Time PCR. Os resultados obtidos sugerem que o gel de metronidazol 25%, aplicado segundo o protocolo estabelecido neste trabalho foi efetivo para a redução dos índices de placa, gengival e do A. actinomycetemcomitans, no entanto, pareceu não atuar sobre a profundidade de sondagem. Os dados obtidos sugerem que, ainda que a ocorrência da bactéria nos indivíduos avaliados tenha sido baixa e a diferença entre o grupo diabéticos e não diabéticos tenha restringido-se apenas à profundidade de sondagem, o gel estudado demonstrou ser promissor na prevenção da doença periodontal em crianças diabéticas, havendo necessidade da realização de mais estudos clínicos randomizados. / Type I Diabetes mellitus (IDDM) is a metabolic disease characterized by deficiency on the insulin production, causing great systemic alterations, including at the oral cavity. Periodontal disease is more aggressive in diabetic patients, even during the childhood, and presents as etiological microorganisms the Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), formerly Actinobacillus actinomycetemcomitans. This microorganisms in association to alterations in the immune and inflammatory answers caused by diabetes, can promote a larger and early alveolar bone resorption even in children. So, the early identification of these aggressors could be extremely useful for the institution of preventive and therapeutic measures in order to inhibit or minimize the noxious effects of IDDM and their consequences in the oral cavity. Aiming this early identification, molecular biology methods of investigation are being used more frequently, pointing out the Real Time Polymerase Chain Reaction (Real-Time PCR), which presents high specificity and sensibility to detect and to quantify microorganisms, even in small number and in different media little explored like saliva. The use of a safe topical antibiotic, such as metronidazole 25% gel, which used in association with dental brushing, could reduce or eliminate A. actinomycetemcomitans, avoiding the progression of the periodontal disease. This study evaluated the presence of A. actinomycetemcomitans before and after the use of metronidazole 25% gel once a day, in substitution to one of the daily teeth brushing. It also evaluated the influence of this treatment protocol on the plaque, gingival and probing depth indexes. Thirty-two children, aging from 3 to 12 years were included in this study and they were divided into two groups: type I diabetic (D) and no diabetic (ND). Real-Time PCR methods were used to evaluate the presence of A. actinomycetemcomitans. The obtained data suggest that metronidazole 25% gel, applied according to the established protocol in this study, effectively reduced plaque and gingival indexes and the number of A. actinomycetemcomitans. However, no influence was observed regarding the probing depth. The obtained data suggest that, although the number of bacteria has been small and the difference between the D and ND groups was not observed only regarding probing depth, the studied antibiotic gel demonstrated to be promising in preventing periodontal disease in diabetic children. More randomized clinical trials are necessary for a final conclusion.

Actinobacillus pleuropneumoniae (App) est l’agent étiologique de la pleuropneumonie porcine, une infection pulmonaire contagieuse chez les porcs. Parmi les nombreux mécanismes de virulence retrouvés chez les bactéries, la formation de biofilms joue souvent un rôle important dans la pathogenèse. Il a été récemment démontré qu’App avait la capacité de former des biofilms in vitro. Dans notre laboratoire, la formation de biofilms par App a été évaluée en microplaques dans différents milieux de culture. Nous avons démontré que la souche de référence de sérotype 1 est capable de former des biofilms. Le but de ce travail est d’identifier des gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. L’objectif de cette étude était de générer une banque de mutants d’App 4074NalR à l’aide du transposon mini-Tn10. Cette banque de 1200 mutants a été criblée à l’aide du modèle in vitro de formation de biofilms en microplaques et en tubes : 24 mutants démontrant une formation de biofilms modifiée par rapport à la souche mère App 4074NalR ont été sélectionnés et identifiés, nous permettant ainsi de localiser le site d’insertion du transposon. Une analyse a permis d’identifier de nouveaux gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. Notre criblage a permis d’identifier 16 gènes connus impliqués dans la formation de biofilms chez App (hns) ou chez d’autres pathogènes (potD2, ptsI, tig and rpmF) mais également de nouveaux gènes impliqués dans la formation de biofilm (APL_0049, APL_0637 and APL_1572). Une caractérisation plus poussée de ces gènes nous permettra d’améliorer la compréhension des mécanismes impliqués dans la formation de biofilm chez App. / A. pleuropneumoniae (App) is the causative agent of porcine pleuropneumonia, a contagious pulmonary infection in swine. Among the numerous virulence mechanisms found in bacteria, the formation of biofilms often plays an important role in pathogenesis. It has been recently demonstrated that App has the ability to form biofilms in vitro. In our laboratory, the formation of biofilms by App has been evaluated in microplates under different growth conditions. We showed that the reference strain of serotype 1 is capable of forming biofilms when cultured in a specific growth medium. The objective of this work is to identifiy genes implicated in the biosynthesis and regulation of biofilm formation in App. The objective of this study was to generate a mutant library of App using the mini-Tn10 transposon. A total of 1200 mutants has been screened with the help of in vitro models for biofilm formation which use microtiter plates or test tubes; 24 mutants exhibited modified biofilm formation when compared to the parental strain 4074NalR. The selection and identification of these mutants allowed the identification of the insertion site of the transposon. Analysis revealed novel genes implicated in biosynthesis and regulation of the biofilm formation in App. Our screen allowed the identification of genes already associated in biofilm formation of App (hns) or other pathogens (potD2, ptsI, tig and rpmF). Genes (APL_0049, APL_0637 and APL_1573) that have not yet been associated with biofilm formation were also identified. Further characterization of the genes mentioned above would permit a greater understanding of the mechanisms implicated in biofilm formation of App.

Ram lamb epididymitis, a serious reproductive disease of sheep, is caused principally by bacteria belonging to the genera Haemophilus and Actinobacillus. Six bacteria were studied: the American Type Culture Collection (ATCC) of Actinobacillus seminis (ATCC 15768), ATCC of Actinobacillus actinomycetemcomitans (ATCC 29522), field isolates of A seminis (86722 and 4101) and field isolates of Actin obacillus- like bacteria (Y136 and D107). The objectives of this study were to quantitate the adhesion of these 6 bacteria to bovine kidney epithelial cells (BKECs) and ram epididymal epithelial cells (REECs), evaluate the effect of rabbit polyclonal antibody prepared against ATCC 15768 (PoAb 15768) on bacterial adherence to BKECs and REECs, and partially characterize the adhesins present on these bacteria. In a bacterial adhesion assay (BAA), strain and species differences were noted. The number of bacteria adhering to each BKEC ranged (rom a low of 4.27 ± 1.00 (Actinobacillus-like 0107) to a high of 31.84 ± 2.00 (A seminis 86722). The number of bacteria adhering to each REEC ranged from a low of 3.05 ± 0.34 (Actinobacill us-like 0107) to a high of 21.61 ± 2.03 (Actinobacillus like Yl36). In a bacterial inhibition assay (BIA), PoAb 15768 inhibited the adhesion of ATCC 15768 to both BKECs and REECs by 5%. This same antiserum inhibited the adhesion of ATCC 29522 to BKECs by 14.5% and to REECs by 22%. The inhibition of A seminis 86722 adherence to BKECs and to REECs was less than 14% and 35%, respectively. For A seminis 4101, Actinobacillus-like Y136, and Actinobacillus-like 0107, PoAb 15768 failed to prevent adhesion to either BKECs or REECs. When the 6 bacteria were analyzed by autoradiography, 2 (Actinobacillus-like 0107) to 8 (ATCC 29522) potential adhesins were identified. However, the pathogenicity has not been firmly established for many Actinobacillus species and Actinobacillus-like bacteria. The potential adhesins identified in this study were not unequivocally confirmed as bacterial adhesins. An in vit ro model may facilitate the recognition of potential adhesins used by Actinobacillus species and Actinobacillus-like bacteria and may eventually lead to the development of an efficacious bacterin to prevent epididymitis in ram lambs at risk.

Succinic acid is a compound used for manufacturing lacquers, resins, and other coating chemicals. It is also used in the food and beverage industry as a flavor additive. It is predominantly manufactured from petrochemicals, but it can also be produced more sustainably by fermentation of sugars from renewable feedstocks (biomass). Bio-based succinic acid has excellent potential for becoming a platform chemical (building block) for commodity and high-value chemicals. In this study, we focused on the production of bio-based succinic acid from the fiber of sweet sorghum (SS), which has a high fermentable sugar content and can be cultivated in a variety of climates and locations around the world. To avoid competition with food feedstocks, we targeted the non-edible ‘bagasse’, which is the fiber part after extracting the juice. Initially, we studied various conditions of pretreating SS bagasse to remove most of the non-fermentable portions and expose the cellulose fibers containing the fermentable sugars (glucose). Concentrated (83%) phosphoric acid was utilized at mild temperatures of 50-80 °C for 30-60 minutes at various bagasse loadings (10-15%) using a partial factorial experimental design. After pretreatment, the biomass was subjected to enzymatic hydrolysis with commercial cellulase enzyme (Cellic® Ctec2) to identify the pretreatment conditions that lead to the highest glucose yield that is critical for the production of succinic acid via fermentation with the bacterium Actinobacillus succinogenes. As the pretreatment temperature and duration increased, the bagasse color changed from light brown to dark brown-black, indicating decomposition, which ranged from 15% to 72%. The pretreatment results were fitted with an empirical model that identified 50 °C for 43 min at 13% solids loading as optimal pretreatment conditions that lead to the highest glucose release from sweet sorghum bagasse. Biomass pretreated at those conditions and subjected to separate enzymatic hydrolysis and fermentation with A. succinogenes yielded almost 18 g/L succinic acid, which represented 90% of the theoretical yield, a very promising performance that warranties further investigation of bio-based succinic acid production from sweet sorghum bagasse, as a more sustainable alternative to succinic acid produced from fossil sources, such as oil.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

Mannose-binding lectin (MBL) and ficolins are complement-activating proteins, and both play an important role in innate immunity by recognizing specific carbohydrate moieties on the surface of wide range of microorganisms. Previous studies have shown that porcine ficolin-α and MBL-A bind to surface polysaccharides of bacteria cultured in suspension, but their interactions with bacteria in biofilm culture have not been studied. The objectives of this thesis were to determine whether porcine plasma ficolin and MBL bind to Actinobacillus pleuropneumoniae in biofilm cultures. APP serotype 5a (APP5a) was used because it produced pronounced biofilm in plastic culture dishes, in comparison with APP5b that was previously reported to bind ficolin in suspension cultures. N-acetylglucosamine (GlcNAc) in the biofilm produced by APP5a was stained with wheat germ agglutinin conjugated with Alexa Fluor-555 and identified by confocal laser scanning microscopy (CLSM). Dispersin B prevented APP5a biofilm formation indicating the requirement of poly N-acetylglucosamine (PNAG) for bacterial cohesion. Bound purified ficolin or ficolin in plasma both were eluted with GlcNAc from APP5a biofilm cultures. To address preferential binding of ficolin-α to biofilm matrix, ficolin-α was eluted with GlcNAc from extracellular polymeric substances (EPS) in supernatant after pelleting the bacteria. Biotinylated-ficolin that retained GlcNAc-binding activity for APP5b planktonic cultures was shown to bind strongly to APP5a biofilm, as detected by fluorescent NeutrAvidin staining and CLSM, but not in the presence of GlcNAc. Further, MBL-A in ficolin-depleted porcine plasma also bound to APP5a biofilm and was eluted with a sugar solution containing GlcNAc, galactose, mannose and glucose. These studies demonstrate that both porcine ficolin-α and MBL-A bind to biofilm cultures of APP5a in a carbohydrate-dependent manner, and suggest that the production of PNAG in biofilm is a binding target for ficolin. / Natural Sciences and Engineering Research Council of Canada (NSERC)

Actinobacillus pleuropneumoniae (App) est l’agent étiologique de la pleuropneumonie porcine, une infection pulmonaire contagieuse chez les porcs. Parmi les nombreux mécanismes de virulence retrouvés chez les bactéries, la formation de biofilms joue souvent un rôle important dans la pathogenèse. Il a été récemment démontré qu’App avait la capacité de former des biofilms in vitro. Dans notre laboratoire, la formation de biofilms par App a été évaluée en microplaques dans différents milieux de culture. Nous avons démontré que la souche de référence de sérotype 1 est capable de former des biofilms. Le but de ce travail est d’identifier des gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. L’objectif de cette étude était de générer une banque de mutants d’App 4074NalR à l’aide du transposon mini-Tn10. Cette banque de 1200 mutants a été criblée à l’aide du modèle in vitro de formation de biofilms en microplaques et en tubes : 24 mutants démontrant une formation de biofilms modifiée par rapport à la souche mère App 4074NalR ont été sélectionnés et identifiés, nous permettant ainsi de localiser le site d’insertion du transposon. Une analyse a permis d’identifier de nouveaux gènes impliqués dans la biosynthèse et dans la régulation de l’expression des biofilms chez App. Notre criblage a permis d’identifier 16 gènes connus impliqués dans la formation de biofilms chez App (hns) ou chez d’autres pathogènes (potD2, ptsI, tig and rpmF) mais également de nouveaux gènes impliqués dans la formation de biofilm (APL_0049, APL_0637 and APL_1572). Une caractérisation plus poussée de ces gènes nous permettra d’améliorer la compréhension des mécanismes impliqués dans la formation de biofilm chez App. / A. pleuropneumoniae (App) is the causative agent of porcine pleuropneumonia, a contagious pulmonary infection in swine. Among the numerous virulence mechanisms found in bacteria, the formation of biofilms often plays an important role in pathogenesis. It has been recently demonstrated that App has the ability to form biofilms in vitro. In our laboratory, the formation of biofilms by App has been evaluated in microplates under different growth conditions. We showed that the reference strain of serotype 1 is capable of forming biofilms when cultured in a specific growth medium. The objective of this work is to identifiy genes implicated in the biosynthesis and regulation of biofilm formation in App. The objective of this study was to generate a mutant library of App using the mini-Tn10 transposon. A total of 1200 mutants has been screened with the help of in vitro models for biofilm formation which use microtiter plates or test tubes; 24 mutants exhibited modified biofilm formation when compared to the parental strain 4074NalR. The selection and identification of these mutants allowed the identification of the insertion site of the transposon. Analysis revealed novel genes implicated in biosynthesis and regulation of the biofilm formation in App. Our screen allowed the identification of genes already associated in biofilm formation of App (hns) or other pathogens (potD2, ptsI, tig and rpmF). Genes (APL_0049, APL_0637 and APL_1573) that have not yet been associated with biofilm formation were also identified. Further characterization of the genes mentioned above would permit a greater understanding of the mechanisms implicated in biofilm formation of App.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [67]-81).