Avec l'augmentation des menaces asymétriques partout sur la planète depuis le début des années 1990s impliquant des armes produisant de forts dommages par rapport à leurs coûts, plusieurs types d'armes biologiques sont devenus une menace d'importance croissante dans le théâtre militaire ainsi que dans le monde civil. Les armes biologiques peuvent êtres facilement dissimulées sur une personne. Elles sont difficilement détectables et leurs déploiements peuvent causer des dommages psychologiques incommensurables sur les populations affectées, assmant un ravage souvent loin au delà de leur emplacement physique. Pour essayer de diminuer l'efficacité de ces armes, des méthodes pour détecter et distinguer en retrait les agents biologiques parmi les autres aérosols naturels présents dans l'atmosphère sont actuellement en développement. Recherche et Développement pour la Défense Canada (RDDC) et le gouvernement du Canada explorent actuellement les LIDARs comme la technologie principale visant la détection à distance d'organismes biologiques. Toutes informations pouvant êtres récoltées à propos des armes biologiques en utilisant cette technologie sont utiles à l'avancement de la cause de la biodétection à distance. Ce rapport présente les résultats d'un projet ayant pom but de décrire les effets de l'âge des bactéries sur leurs propriétés spectrales de fluorescence en utilisant la technologie LIDAR. Les expériences ont été exécutées dans une chambre hermétique LIDAR de laboratoire sur des simulants d'agents biologiques connues, trois desquelles (2 souches de Bacillus subtilis var. niger identifiées comme BGnew et BGold et une souche de Bacillus thuringiensis identifié comme BT) étant des simulants de la bactérie Bacillus anthracis (l'anthrax), sans doute l'agent biologique le plus probable à être utilisé, et l'autre (Erwinia herbicola identifié comme EH) étant un simulant de Yersinia pestis (la peste). Les bactéries étaient mélangées avec leur milieu de culture, soit le ± Tryptic soy broth ¿ (TSB). Bien que plus de tests soient nécessaires afin de pouvoir finaliser des conclusions plus robustes, certaines tendances ont été observées. Le mélange de Erwinia herbicola a montré une fluorescence induite ayant une tendance vers les courtes longueurs d'ondes lorsque la culture était plus âgée. Le mélange de Bacillus thuringiensis (BT) a démontré une petite tendance vers les longues longueurs d'ondes avec l'âge. Les deux différentes souches de Bacillus globigii ont montrées des fluorescences induites ayant des tendances vers les courtes longueurs d'ondes après un mois et vers les longues longueurs d'ondes après trois mois. En bref, la signature de fluorescence semble dépendre de l'âge du mélange bactérie-bouillon de culture bien que cet effet varie en fonction du type mélange. Certains mélanges étaient plus efficaces à produire de la fluorescence que d'autres. Le mélange de BGnew-ljour a démontré la plus haute efficacité de fluorescence tandis que BG_old-3mois, BGold-ljour et EH-ljour ont démontrés les plus faibles. Nos résultats démontrent que la signature spectrale d'une bactérie est influencée par le temps d'incubation dans son milieu de culture. Cette information est très importante lors de l'élaboration de banques de signatures standardisées servant à la classification bactérienne à distance dans le cadre d'intervention sur le terrain. Dans des contextes militaires et de sécurité publique, cette information serait aussi un outil important pour retrouver l'origine des agents biologiques (ex: s'ils parviennent d'un labo ou d'un lieu d'entreposage).

Dentre as 18 espécies do gênero Yersinia, as espécies Y. enterocolitica, Y. pseudotuberculosis e Y. pestis foram extensivamente caracterizadas em diversos aspectos como ecologia, epidemiologia e mecanismos de patogenicidade. Sete das 15 espécies restantes (Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollaretii e Y. rohdei), usualmente conhecidas como Y. enterocolitica-like, até o momento, não tiveram seu potencial patogênico caracterizado e são, geralmente, consideradas não-patogênicas. Entretanto, dados da literatura sugerem que algumas dessas espécies possam causar doença. Esses dados estimularam o surgimento de questões sobre os mecanismos pelos quais as espécies de Y. enterocolitica-like possam interagir com as células do hospedeiros e causar doenças. Esse projeto teve como principal objetivo caracterizar o potencial patogênico de linhagens de Y. enterocolitica-like, especificamente das espécies Y. frederiksenii, Y. kristensenii e Y. intermedia. No presente trabalho, o potencial patogênico de 118 linhagens de Y. enterocolitica-like (50 Y. frederiksenii, 55 Y. intermedia e 13 Y. kristensenii) foi avaliado pela pesquisa da presença dos genes relacionados à virulência ail, fepA, fepD, fes, hreP, myfA, tccC, ystA, ystB e virF por PCR. Além disso, a habilidade de algumas linhagens de Yersinia de aderir e invadir células Caco-2 e HEp-2 após diferentes períodos de incubação, bem como, de sobreviver no interior de macrófagos humanos U937 foi testada. Aspectos morfológicos da adesão bacteriana foram visualizados por microscopia eletrônica. Finalmente, a presença de possíveis novos mecanismos de virulência foi avaliada a partir do sequenciamento de RNA de uma linhagem de Y. enterocolitica-like. As linhagens estudadas apresentaram os seguintes genes: Y. frederiksenii, fepA (44%), fes (44%) e ystB (18%); Y. intermedia, ail (53%), fepA (35%), fepD (2%), fes (97%), hreP (2%), ystB (2%) e tccC (35%); e Y. kristensenii, ail (62%), ystB (23%), fepA (77%), fepD (54%), fes (54%) e hreP (54%). De modo geral, as linhagens de Y. enterocolitica-like tiveram a habilidade de aderir e invadir células Caco-2 e HEp-2 inferior à da linhagem altamente patogênica Y. enterocolitica 8081. Contudo, Y. kristensenii FCF 410 e Y. frederiksenii FCF 461 apresentaram elevado potencial de invasão a células Caco-2 após cinco dias de pré-incubação, os quais foram 45 e 7,2 vezes maiores do que o controle Y. enterocolitica 8081, respectivamente, porém, o gene ail não foi detectado nessas linhagens. O ensaio de sobrevivência em macrófagos humanos U937 ii mostrou que as linhagens de Y. frederiksenii FCF 461 (40,0%) e Y. frederiksenii FCF 379 (24,6%) tiveram porcentagens de sobrevivência superior à de Y. enterocolitica 8081 (13,4%). Todavia, linhagens de Y. intermedia e Y. kristensenii apresentaram uma capacidade reduzida de sobreviver em macrófagos. A microscopia eletrônica de varredura mostrou as bactérias em contato com a filipódia celular. As bactérias foram distribuídas tanto individualmente quanto em pequenos aglomerados. Portanto, podemos concluir que a presença dos genes relacionados à virulência encontrados nas Y. enterocolitica-like estudadas indicou o possível potencial patogênico de algumas dessas linhagens. Os ensaios de adesão e invasão a células de mamíferos sugerem que a patogenicidade de Y. kristensenii e Y. frederiksenii possa ser linhagem-dependente. O ensaio de sobrevivência em macrófagos humanos U937 evidenciou o potencial patogênico de algumas linhagens de Y. frederiksenii. Em conjunto, os resultados obtidos sugerem a existência de mecanismos de virulência alternativos aos mecanismos clássicos descritos para Y. enterocolitica patogênica. Contudo, a presença de possíveis novos mecanismos de virulência não pode ser verificada, uma vez que a plataforma 454 GS Junior (Roche) não se mostrou adequada para a realização de sequenciamento de RNA de amostras provenientes de interações com células devido à baixa cobertura obtida. / Among the 18 species of the Yersinia genus, Y. enterocolitica, Y. pseudotuberculosis and Y. pestis were extensively characterized in different subjects as ecology, epidemiology and pathogenicity mechanisms. Seven among the remaining 15 species (Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollaretii and Y. rohdei), often called Y. enterocolitica-like have not their pathogenic potential characterized and are usually considered to be nonpathogenic. However, literature data suggest that some of these species can cause diseases. These data stimulate the upsurge of questions about the mechanisms of which Y. enterocolitica-like species may interact with host cells and cause diseases. The main objective of this preject was to characterize the pathogenic potential of Y. enterocolitica-like strains, specifically of the species Y. frederiksenii, Y. kristensenii and Y. intermedia. This work evaluated the pathogenic potential of 118 Y. enterocolitica-like strains (50 Y. frederiksenii, 55 Y. intermedia and 13 Y. kristensenii) searching for the presence of the virulence-related genes ail, fepA, fepD, fes, hreP, myfA, tccC, ystA, ystB and virF by PCR. Besides, Yersinia strains ability of adhesion and invasion to Caco-2 and HEp-2 cells after different pre-incubation periods, and its survival within human macrophages U937 were tested. Morphologic aspects of bacterial adhesion were observed by scanning electronic microscopy. Finally, the presence of new possible virulence mechanisms was evaluated through RNA sequencing of one Y. enterocolitica-like strain. The studied strains showed the following genes: Y. frederiksenii, fepA (44%), fes (44%) and ystB (18%); Y. intermedia, ail (53%), fepA (35%), fepD (2%), fes (97%), hreP (2%), ystB (2%) and tccC (35%); and Y. kristensenii, ail (62%), ystB (23%), fepA (77%), fepD (54%), fes (54%) and hreP (54%). Usually Y. enterocolitica-like strains presented less ability of adhere and invade Caco-2 and HEp-2 cells than the highly pathogenic strain Y. enterocolitica 8081. On the other hand, Y. kristensenii FCF 410 and Y. frederiksenii FCF 461 showed high potential of invasion in Caco-2 cells after 5 days of pre-incubation, which were 45 and 7.2 times higher than the control Y. enterocolitica 8081 respectively, but ail gene was not found in these strains. Survival assay in human macrophages U937 showed that Y. frederiksenii FCF 461 (40.0%) and Y. frederiksenii FCF 379 (24.6%) strains presented survival percentages higher than Y. enterocolitica 8081 (13.4%). However, Y. intermedia and Y. iv kristensenii strains showed a reduced capability of surviving in macrophages. Scanning electron microscopy showed bacteria at the surface in contact with the cellular filopodia. The bacteria were distributed either individually or in small clumps. Therefore, it may be concluded that the presence of virulence-related genes in some of the Y. enterocolitica-like strains indicated their possible pathogenic potential. Mammal cells adhesion and invasion assays suggest that the pathogenicity of Y. kristensenii and Y. frederiksenii may be strain-dependent. Human macrophages U937 surviving assay highlighted the pathogenic potential of some Y. frederiksenii strains. Together, the results suggest the existence of alternative virulence mechanisms other than the classical mechanisms described for pathogenic Y. enterocolitica. However, we could not verify the presence of possible new virulence mechanisms because 454 GS Junior (Roche) platform was not suitable for RNA sequencing of strains from cells interaction due its low coverage obtained.

Génératrice depuis le VIe siècle de notre ère de crises épidémiques récurrentes en Occident, la peste a profondémentmarqué l’histoire des sociétés européennes, tant sur le plan biologique que culturel, économique et politique. Sil’histoire des épidémies qu’elle a engendrées est aujourd’hui relativement bien connue, un certain nombre de questionssur ses caractéristiques épidémiologiques passées demeurent pour partie irrésolues. En particulier, le caractère sélectifou non de la mortalité par peste à l’égard de l’âge, du sexe et de l’état de santé préexistant des individus faitactuellement débat. À partir d’une approche anthropobiologique, le présent travail se propose de contribuer à cettediscussion. Il livre les résultats de l’étude d’un corpus de 1090 squelettes provenant, d’une part, de quatre sitesd’inhumation de pestiférés de la fin du Moyen Âge et du début de l’époque moderne et, d’autre part, de deuxcimetières paroissiaux médiévaux utilisés hors contexte épidémique. Cette étude révèle en premier lieu l’existenced’une signature démographique commune aux séries en lien avec la peste. Leur composition par âge et par sexe,distincte de celle caractérisant la mortalité naturelle, est au contraire en adéquation avec la structure théorique d’unepopulation vivante préindustrielle. L’examen de divers indicateurs de stress suggèrent par ailleurs que les victimes dela peste jouissaient, à la veille de leur décès, d’un meilleur état de santé que les individus morts en temps normal. Lesrésultats obtenus concourent à démontrer que les facteurs causals de ces lésions squelettiques, d’accoutuméresponsables d’une diminution des chances de survie, n’eurent au contraire qu’une influence mineure, si ce n’est nulle,sur le risque de mourir de l’infection à Yersinia pestis. Ce travail livre in fine un faisceau d’arguments convergents quitendent à prouver que les épidémies de peste anciennes furent à l’origine d’une mortalité non sélective, la maladiefrappant indistinctement les individus des deux sexes, de tous âges et de toutes conditions sanitaires. / From the 6th century onwards, plague caused recurring mortality crises in the Western world. Such epidemics hadprofound biological, cultural, economic and political impacts on European societies. Some aspects of the history ofplague epidemics are currently well known, but many questions remain unanswered, such as the preciseepidemiological pattern of the disease in ancient times. It is unclear whether plague killed people indiscriminately orwhether this disease was selective with respect to age, sex and health. This research contributes to this debate.It consists of an anthropological and paleopathological study of skeletal remains of 1090 individuals, including plaguevictims from four medieval and post-medieval burial grounds, and individuals from two parochial cemeteries in useduring periods of normal mortality. Results from the four plague-related assemblages reveal a peculiar demographicsignature. Age and sex distribution differs clearly from what is expected in non-epidemic periods, when it is shown tocorrespond closely to the demographic structure of the living population. Moreover, the study of various non-specificskeletal stress markers shows that plague victims were in a better health before they passed away than people who diedin non-epidemic periods. The results demonstrate that individuals who suffered stress and disease had a reducedchance of survival in non-epidemic periods, whereas they were not at a higher risk to die during plague epidemics.This study provides evidence that plague was not selective, and that it killed regardless of sex, age, and pre-existing health.

Dentre as 18 espécies do gênero Yersinia, as espécies Y. enterocolitica, Y. pseudotuberculosis e Y. pestis foram extensivamente caracterizadas em diversos aspectos como ecologia, epidemiologia e mecanismos de patogenicidade. Sete das 15 espécies restantes (Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollaretii e Y. rohdei), usualmente conhecidas como Y. enterocolitica-like, até o momento, não tiveram seu potencial patogênico caracterizado e são, geralmente, consideradas não-patogênicas. Entretanto, dados da literatura sugerem que algumas dessas espécies possam causar doença. Esses dados estimularam o surgimento de questões sobre os mecanismos pelos quais as espécies de Y. enterocolitica-like possam interagir com as células do hospedeiros e causar doenças. Esse projeto teve como principal objetivo caracterizar o potencial patogênico de linhagens de Y. enterocolitica-like, especificamente das espécies Y. frederiksenii, Y. kristensenii e Y. intermedia. No presente trabalho, o potencial patogênico de 118 linhagens de Y. enterocolitica-like (50 Y. frederiksenii, 55 Y. intermedia e 13 Y. kristensenii) foi avaliado pela pesquisa da presença dos genes relacionados à virulência ail, fepA, fepD, fes, hreP, myfA, tccC, ystA, ystB e virF por PCR. Além disso, a habilidade de algumas linhagens de Yersinia de aderir e invadir células Caco-2 e HEp-2 após diferentes períodos de incubação, bem como, de sobreviver no interior de macrófagos humanos U937 foi testada. Aspectos morfológicos da adesão bacteriana foram visualizados por microscopia eletrônica. Finalmente, a presença de possíveis novos mecanismos de virulência foi avaliada a partir do sequenciamento de RNA de uma linhagem de Y. enterocolitica-like. As linhagens estudadas apresentaram os seguintes genes: Y. frederiksenii, fepA (44%), fes (44%) e ystB (18%); Y. intermedia, ail (53%), fepA (35%), fepD (2%), fes (97%), hreP (2%), ystB (2%) e tccC (35%); e Y. kristensenii, ail (62%), ystB (23%), fepA (77%), fepD (54%), fes (54%) e hreP (54%). De modo geral, as linhagens de Y. enterocolitica-like tiveram a habilidade de aderir e invadir células Caco-2 e HEp-2 inferior à da linhagem altamente patogênica Y. enterocolitica 8081. Contudo, Y. kristensenii FCF 410 e Y. frederiksenii FCF 461 apresentaram elevado potencial de invasão a células Caco-2 após cinco dias de pré-incubação, os quais foram 45 e 7,2 vezes maiores do que o controle Y. enterocolitica 8081, respectivamente, porém, o gene ail não foi detectado nessas linhagens. O ensaio de sobrevivência em macrófagos humanos U937 ii mostrou que as linhagens de Y. frederiksenii FCF 461 (40,0%) e Y. frederiksenii FCF 379 (24,6%) tiveram porcentagens de sobrevivência superior à de Y. enterocolitica 8081 (13,4%). Todavia, linhagens de Y. intermedia e Y. kristensenii apresentaram uma capacidade reduzida de sobreviver em macrófagos. A microscopia eletrônica de varredura mostrou as bactérias em contato com a filipódia celular. As bactérias foram distribuídas tanto individualmente quanto em pequenos aglomerados. Portanto, podemos concluir que a presença dos genes relacionados à virulência encontrados nas Y. enterocolitica-like estudadas indicou o possível potencial patogênico de algumas dessas linhagens. Os ensaios de adesão e invasão a células de mamíferos sugerem que a patogenicidade de Y. kristensenii e Y. frederiksenii possa ser linhagem-dependente. O ensaio de sobrevivência em macrófagos humanos U937 evidenciou o potencial patogênico de algumas linhagens de Y. frederiksenii. Em conjunto, os resultados obtidos sugerem a existência de mecanismos de virulência alternativos aos mecanismos clássicos descritos para Y. enterocolitica patogênica. Contudo, a presença de possíveis novos mecanismos de virulência não pode ser verificada, uma vez que a plataforma 454 GS Junior (Roche) não se mostrou adequada para a realização de sequenciamento de RNA de amostras provenientes de interações com células devido à baixa cobertura obtida. / Among the 18 species of the Yersinia genus, Y. enterocolitica, Y. pseudotuberculosis and Y. pestis were extensively characterized in different subjects as ecology, epidemiology and pathogenicity mechanisms. Seven among the remaining 15 species (Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollaretii and Y. rohdei), often called Y. enterocolitica-like have not their pathogenic potential characterized and are usually considered to be nonpathogenic. However, literature data suggest that some of these species can cause diseases. These data stimulate the upsurge of questions about the mechanisms of which Y. enterocolitica-like species may interact with host cells and cause diseases. The main objective of this preject was to characterize the pathogenic potential of Y. enterocolitica-like strains, specifically of the species Y. frederiksenii, Y. kristensenii and Y. intermedia. This work evaluated the pathogenic potential of 118 Y. enterocolitica-like strains (50 Y. frederiksenii, 55 Y. intermedia and 13 Y. kristensenii) searching for the presence of the virulence-related genes ail, fepA, fepD, fes, hreP, myfA, tccC, ystA, ystB and virF by PCR. Besides, Yersinia strains ability of adhesion and invasion to Caco-2 and HEp-2 cells after different pre-incubation periods, and its survival within human macrophages U937 were tested. Morphologic aspects of bacterial adhesion were observed by scanning electronic microscopy. Finally, the presence of new possible virulence mechanisms was evaluated through RNA sequencing of one Y. enterocolitica-like strain. The studied strains showed the following genes: Y. frederiksenii, fepA (44%), fes (44%) and ystB (18%); Y. intermedia, ail (53%), fepA (35%), fepD (2%), fes (97%), hreP (2%), ystB (2%) and tccC (35%); and Y. kristensenii, ail (62%), ystB (23%), fepA (77%), fepD (54%), fes (54%) and hreP (54%). Usually Y. enterocolitica-like strains presented less ability of adhere and invade Caco-2 and HEp-2 cells than the highly pathogenic strain Y. enterocolitica 8081. On the other hand, Y. kristensenii FCF 410 and Y. frederiksenii FCF 461 showed high potential of invasion in Caco-2 cells after 5 days of pre-incubation, which were 45 and 7.2 times higher than the control Y. enterocolitica 8081 respectively, but ail gene was not found in these strains. Survival assay in human macrophages U937 showed that Y. frederiksenii FCF 461 (40.0%) and Y. frederiksenii FCF 379 (24.6%) strains presented survival percentages higher than Y. enterocolitica 8081 (13.4%). However, Y. intermedia and Y. iv kristensenii strains showed a reduced capability of surviving in macrophages. Scanning electron microscopy showed bacteria at the surface in contact with the cellular filopodia. The bacteria were distributed either individually or in small clumps. Therefore, it may be concluded that the presence of virulence-related genes in some of the Y. enterocolitica-like strains indicated their possible pathogenic potential. Mammal cells adhesion and invasion assays suggest that the pathogenicity of Y. kristensenii and Y. frederiksenii may be strain-dependent. Human macrophages U937 surviving assay highlighted the pathogenic potential of some Y. frederiksenii strains. Together, the results suggest the existence of alternative virulence mechanisms other than the classical mechanisms described for pathogenic Y. enterocolitica. However, we could not verify the presence of possible new virulence mechanisms because 454 GS Junior (Roche) platform was not suitable for RNA sequencing of strains from cells interaction due its low coverage obtained.

Yersinia pestis (Y. pestis), as the causative agent of plague, has caused deaths estimated to more than 200 million people in three historical plague pandemics, including the infamous Black Death in medieval Europe. Although infection with Yersinia pestis can mostly be limited by antibiotics and only 2000-5000 cases are observed worldwide each year, this bacterium is still a concern for bioterrorism and recognized as a category A select agent by the Centers for Disease Control and Prevention (CDC). The investigation into the host-pathogen interactions during Y. pestis infection is important to advance and broaden our knowledge about plague pathogenesis for the development of better vaccines and treatments. Y. pestis is an expert at evading innate immune surveillance through multiple strategies, several mediated by its type three secretion system (T3SS). It is known that the bacterium induces rapid and robust cell death in host macrophages and dendritic cells. Although the T3SS effector YopJ has been determined to be the factor inducing cytotoxicity, the specific host cellular pathways which are targeted by YopJ and responsible for cell death remain poorly defined. This thesis research has established the critical roles of caspase-8 and RIP kinases in Y. pestis-induced macrophage cell death. Y. pestis-induced cytotoxicity is completely inhibited in RIP1-/- or RIP3-/-caspase-8-/- macrophages or by specific chemical inhibitors. Strikingly, this work also indicates that macrophages deficient in either RIP1, or caspase-8 and RIP3, have significantly reduced infection-induced production of IL-1β, IL-18, TNFα and IL-6 cytokines; impaired activation of NF-κB signaling pathway and greatly compromised caspase-1 processing; all of which are critical for innate immune responses and contribute to fight against pathogen infection. Y. pestis infection causes severe and often rapid fatal disease before the development of adaptive immunity to the V bacterium, thus the innate immune responses are critical to control Y. pestis infection. Our group has previously established the important roles of key molecules of the innate immune system: TLR4, MyD88, NLRP12, NLRP3, IL-18 and IL-1β, in host responses against Y. pestis and attenuated strains. Yersinia has proven to be a good model for evaluating the innate immune responses during bacterial infection. Using this model, the role of caspase-8 and RIP3 in counteracting bacterial infection has been determined in this thesis work. Mice deficient in caspase-8 and RIP3 are very susceptible to Y. pestis infection and display reduced levels of pro-inflammatory cytokines in spleen and serum, and decreased myeloid cell death. Thus, both in vitro and in vivo results indicate that caspase-8 and RIP kinases are key regulators of macrophage cell death, NF-κB and caspase-1 activation in Yersinia infection. This thesis work defines novel roles for caspase-8 and RIP kinases as the central components in innate immune responses against Y. pestis infection, and provides further insights to the host-pathogen interaction during bacterial challenge.

Outbreaks of food-born illness associated with the consumption of unpasteurized juice and apple cider have resulted in a rule published by the U.S. Food and Drug Administration (FDA) in order to improve the safety of juice products. The rule (21 CFR120) requires manufacturers of juice products to develop a Hazard Analysis and Critical Control Point (HACCP) plan and to achieve a 5-log reduction in the number of the most resistant pathogens. Ultraviolet (UV) disinfection is one of the promising methods to reach this 5-log reduction of pathogens. The absorption coefficients of juices typically vary from 10 to 40 1/cm and can be even higher depending on brand and processing conditions. Thin film reactors consisting of two concentric cylinders are suitable for inactivating pathogens in juices. When the two concentric cylinders are fixed, the flow pattern in the gap can be laminar Poiseuille flow or turbulent flow depending on flow rates. If the inner cylinder is rotating, and the rotating speed of the inner cylinder exceeds a certain value, the flow pattern can be either laminar or turbulent Taylor-Couette flow. UV disinfection between concentric cylinders in laminar Poiseuille flow, turbulent flow and both laminar and turbulent Taylor-Couette flow was investigated experimentally and numerically. This is the first systematic study done on UV disinfection between concentric cylinders in all three flow patterns. The present work provides new experimental data for pathogen inactivation in each of the three flow patterns. In addition, the present study constitutes the first systematic numerical CFD predictions of expected inactivation levels. Proper operating parameters and optimum gap widths for different flow patterns are suggested. It is concluded that laminar Poiseuille flow provides inferior (small) inactivation levels while laminar Taylor-Couette flow provides superior (large) inactivation levels. The relative inactivation levels are: laminar Poiseuille flow < turbulent flow < laminar Taylor-Couette flow.

Of the eleven species of bacteria that comprise the genus Yersinia of the family Enterobacteriaceae, three species are pathogenic for humans. Yersinia pseudotuberculosis and Yersinia enterocolitica usually cause a mild, self-limiting mesenteric lymphadenitis or ileitis. Yersinia pestis causes a highly invasive often fatal disease known as plague. All three elaborate a type three secretion system that is essential for virulence and encoded on closely related plasmids. In Y. pestis, all the effectors, structural components and chaperones are encoded on the 70kb plasmid, pCD1. Of these, LcrV from Y. enterocolitica has been implicated in playing an immunosuppressive role through its interaction with host Toll-like receptor 2 (TLR2) and induction of IL-10. Through expression and purification of recombinant LcrV from Escherichia coliwe show that only high molecular weight species of rLcrV are able to stimulate TLR2. In a highly sensitive subcutaneous mouse infection model we demonstrate no difference in the time to death between TLR2-sufficient or deficient mice. Analysis of cytokine levels between these two genotypes also shows no significant difference between splenic IL-10 and IL-6 or levels of bacteria. We conclusively show that this interaction, if it does occur, plays no significant role in vivo. In a separate set of experiments, we also determined that the expression of F1, a peptide shown to be responsible for 37°C-dependent inhibition of invasion by Y. pestis in vitro, was significantly decreased under high oxygen conditions. This led us to re-examine the invasion phenotype both in vitro and in vivo. These results give new insights into virulence gene expression in Y. pestis by environmental cues other than temperature.

Die Infektion mit Salmonella und Yersinia (Y.) enterocolitica über Produkte tierischen Ursprungs stellt nach wie vor ein ungelöstes Problem des gesundheitlichen Verbraucherschutzes dar. Will man diese Zoonoseerreger aus der Lebensmittelkette fernhalten, sind moderne und gut validierte Nachweissysteme erforderlich. Eine Infektion von Schweinen erfolgt überwiegend im Mastbetrieb mit Infektionsdosen, die nur zu einer milden klinischen Symptomatik führen. In den meisten Fällen überstehen die Tiere die Infektion mit Salmonella und Y. enterocolitica und werden zu klinisch inapparenten Keimträgern. Solche Schweine stellen ein Reservoir für die Infektion anderer Tiere und für den Eintrag in die Lebensmittelkette dar. Im Rahmen dieser Arbeit wurden zwei PCR-Methoden zum spezifischen Nachweis von Salmonella und Y. enterocolitica im Schlachtschwein entwickelt und anhand von Probenmaterial aus eigens dafür durchgeführten Infektionsversuchen mit S. Typhimuirum und Y. enterocolitica evaluiert. Beide Methoden mussten sich am diagnostischen Goldstandard für den jeweiligen Erreger messen lassen. Für Salmonella Typhimurium wurde die ISO-Norm 6579 und für Y. enterocolitica die ISO-Norm 10273 zum kulturellen Nachweis ausgewählt. Es konnte eine neue PCR-Methodik zum Salmonella-Nachweis in 14 verschiedenen Gewebeproben etabliert werden, die im Vergleich zum kulturellen Nachweis nach ISO 6579 eine Sensitivität von 100 % und eine Spezifität von 96 % aufweist und die Zeitspanne bis zum spezifischen Nachweis des Erregers um mindestens 24 Stunden reduziert. Die Untersuchungen erfolgten anhand von 420 Gewebeproben aus persistent infizierten Schweinen aus Infektionsversuchen mit S. Typhimurium DT104. Dieses neu entwickelte und validierte PCR-Verfahren wurde mit einem bereits etablierten PCR-Nachweissystem nach RAHN et al. (1992) - wie in der DIN 10135 angegeben - verglichen. Beide PCR-Methoden basieren auf dem invA-Virulenzgen von S. Typhimurium. Im Infektionsversuch konnten zwei Gewebeproben (Caecum und Lnn. Ileocolici) bestimmt werden, durch deren Kombination man mit beiden Nachweismethoden 96 % (23 von 24 Tieren) aller im Versuch infizierten Schweine als Salmonella positiv identifizieren konnte. Erstmals gelang in dieser Arbeit der Nachweis des yopT-Gens bei plasmidtragenden Y. pseudotuberculosis-Stämmen sowie die Bestimmung der Sequenz (European Bioinformatics Institute, Accession-Number: AJ304833). Das yopT-Gen kodiert für ein 35,5 kDa großes Effektor-Protein, das einen zytotoxischen Effekt auf HELA-Zellen und Makrophagen besitzt. Durch den Nachweis des yopT-Gens bei Y. pseudotuberculosis-Stämmen war es erstmals möglich, eine für Y. enterocolitica spezifische, auf dem yopT-Gen des Virulenzplasmids basierende PCR-Methode zu etablieren, die auch die Diskriminierung von Y. pseudotuberculosis-Isolaten gestattet. In einem weiteren Infektionsversuch konnte gezeigt werden, dass es die auf dem yopT-Gen von Y. enterocolitica basierende PCR-Methode erlaubt, Carrier-Tiere mit hoher Sensitivität (100 %) und Spezifität (87 %) innerhalb von 56 Stunden in lymphatischen Geweben zu identifizieren. Besonders geeignet für den Nachweis mit der ISO 10273 und dem neu etablierten yopT PCR-Verfahren waren das Ileum und die Lnn. ileocolici. In dieser Arbeit ist der Versuch gelungen, die Diagnostik für zwei der drei wichtigsten beim Schwein vorkommenden humanen Enteritiserreger zu standardisieren, indem Kombinationen aus Gewebeproben bestimmt wurden, die sowohl für den Nachweis mit der jeweiligen Goldstandard-Methode als auch mit den schnelleren und sensitiveren PCR-Methoden geeignet sind. Die Ergebnisse dieser Arbeit tragen zu einer deutlichen Verbesserung der Diagnostik von Salmonella und Y. enterocolitica beim Schlachtschwein bei. Es bleibt zu hoffen, dass somit der Eintrag dieser Zoonoseerreger in die Lebensmittelkette reduziert und der Verbraucherschutz auf diesem Gebiet beträchtlich verbessert werden kann. / The infection with Salmonella and Yersinia (Y.) enterocolitica through foodstuff from slaughter pigs is one of the major problems of hygienic consumer protection. To avoid the contamination of products from pig industry modern and well validated bacteriological identification systems are necessary. An infection predominantly occurs in the fattening pens, showing mild clinical symtoms only. The majority of infected pigs overcome the infection with Salmonella and Y. enterocolitica and become clinically inapparent carrier pigs. Those pigs are a reservoir for the contamination of other animals and pork products. In the context of this work two PCR-assays for the specific detection of Salmonella and Y. enterocolitica have been developed and validated on the basis of tissue samples from experimentally infected pigs. Both methods have been compared with the classical bacterial culture. Two international standards were used for bacterial detection: ISO 6579 for S. Typhimurium and ISO 10273 for pathogenic Y. enterocolitica. It was possible to establish a new PCR-assay for the specific detection of Salmonella in 14 different tissues of experimentally infected pigs. In comparison to the standard ISO 6579 a sensitivity of 100 % and a specificity of 96 % were calculated for the PCR-assay. The investigations were carried out with 420 tissue samples of persistently infected pigs that have been experimentally infected with S. Typhimurium. By using the PCR-method for the detection of Salmonella in positive tissue samples, the detection-time could be reduced around 24 hours. The new PCR-assay developed and validated in this work, was compared with the PCR-method described in DIN 10135, which is based on the studies of RAHN et al. (1992). Both methods were based on the invA-virulence gene of S. Typhimurium. A combination of samples from ileocolic lymph node and caecum was particularly suitable for the detection of 96 % of the experimentally infected pigs (23 off 24 animals) with the PCR-assay and the culture method. In this study, the yopT-gene was proved for the first time to be present in plasmid bearing Y. pseudotuberculosis-Isolates, and the nucleotid sequence was determined (European Bioinformatics Institute, Accession-Number: AJ304833). YopT encodes a 35.5 kDa effector protein (YopT), which induces a cytotoxic effect in HeLa cells and macrophages. This finding was used to develop a specific PCR-assay for the detection of pathogenic Y. enterocolicica strains and the discrimination from pathogenic Y. pseudotuberculosis strains. Embedded in an experimental Y. enterocolitica-infection-model in swine, it was shown that the yopT PCR-assay is suitable for the detection of pathogenic Y. enterocolitica in lymphatic tissue of persistently infected pigs. The yopT PCR-method shows a sensitivity of 100 % and a specificity of 87 % in lymphatic tissue. By the use of the PCR-assay, the detection of Y. enterocolitica was possible within 56 hours. A combination of specimens from the ileum and ileocolic lymph nodes was most suitable for the detection of pathogenic Y. enterocolitica in slaughter pigs with the ISO-Standard 10273 and the yopT PCR. This investigation succeeded in standardizing the identification of two of the three most important zoonotic agents for human enteric disease. The standardization was achived by the use of a combination of samples suitable for the identification with both, the “Goldstandard” and the specific and rapid PCR-method. The results of this work offer a better identification of Salmonella and Y. enterocolitica in slaughter pigs in the future. Based on these facts it is possible to avoid contamination of food products from slaughter pigs and to improve the hygienic consumer protection considerably.

Ausgehend von dem geschichtlichen Pestausbruch des 14. Jahrhunderts in Europa werden 350 Jahre Geschichte der Pest als Pandemie, Epidemie, Endemie und Pest der Kinder in Deutschland, dem Kurfürstentum Sachsen und der Residenzstadt Dresden mit dem Amt Radeberg untersucht und dargestellt. Wichtig war dabei die Herausstellung, dass Pandemien durchaus die Welt verändern und der „Krieg der Mikroben, als sogenannte „Unsichtbare Feinde”, Zivilisationen immer wieder mit ihren eingefahrenen Strukturen zum Wanken bringen können. Dokumentiert wird die Sterbensgefahr durch die Pestausbrüche und deren Überlieferung in Dresdner Akten und dem Amt Radeberg, welches auch zum Fluchtort der Kurfürsten bei Pestgefahr wurde. Herausgearbeitet wurde ebenfalls die stetige Pestgefahr, die als Bedrohung durch das nahe Grenzgebiet von Böhmen ausging, durch Glaubensflüchtlinge beziehungsweise mit dem Handel auf dem Wasser- oder Landwege nach Sachsen gebracht oder durch die Heerscharen des Dreißigjährigen Krieges verbreitet wurde. Nachdem die Pest 1682 offiziell als „überwunden“ erklärt wurde, traten bis in die Jetztzeit immer wieder epidemische oder endemische Pestausbrüche auf, die zeigen - die Pest lebt weiter…