Objectives: Fusobacteria are prominent participants in the maturation of subgingival dental plaque biofilms in humans. A number of various species belonging to the Fusobacterium genus have been recovered from the subgingival microbiota of chronic periodontitis patients. However, conventional Fusobacterium species identification is labor-intensive, time-consuming, and complicated by shortcomings in phenotypic-based classification schemes, where many fusobacteria display overlapping and non-distinguishing morphologic features and biochemical properties. In addition, molecular identification of fusobacteria is plagued with difficulties of validating the specificity of nucleic acid probes and primers to various Fusobacterium species that have closely-related interspecies genetic profiles. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and its associated analytic software, was recently approved for clinical microbiology diagnostic use by the United States Food and Drug Administration. MALDI-TOF mass spectrometry has the potential to rapidly identify cultivable clinical isolates to a species level for 4,613 different bacterial species based on mass spectra of their bacterial protein profiles, including many Fusobacterium species. The purpose of this study was to use MALDI-TOF mass spectrometry to rapidly identify the patient distribution of fusobacteria isolated from the subgingival microbiota of chronic periodontitis patients. Methods: A total of 34 chronic periodontitis patients provided 96 fresh subgingival cultivable fusobacteria isolates (one to seven isolates per patient), which were presumptively identified by their chartreuse-positive colony autofluorescence under long-wave ultraviolet light on anaerobically-incubated, non-selective, enriched Brucella blood agar primary isolation plates. Each of the presumptive fusobacteria clinical isolates were subjected to MALDI-TOF mass spectrometry analysis using a bench top mass spectrometer, Bruker FlexControl 3.0 software, and MALDI Biotyper 3.1 software (Bruker Daltonics, Billerica, MA, USA), which contains mass spectra for a variety of fusobacteria in its reference library of bacterial protein profiles. Each clinical isolate underwent on-target plate formic acid protein extraction, and was taxonomically classified with MALDI-TOF mass spectrometry within an approximately 30-45 minute time period from the point of colony harvesting from primary isolation culture plates. A MALDI Biotyper log score of equal to or larger than 1.7 was required for reliable taxonomic classification of the clinical fusobacteria isolates. Results: A majority (58.8%) of the chronic periodontitis patients yielded two or three different species of subgingival Fusobacterium on non-selective enriched Brucella blood agar primary isolation plates. Fusobacterium naviforme was identified by MALDI-TOF mass spectrometry analysis in 14 (41.2%) chronic periodontitis study patients, Fusobacterium nucleatum subspecies vincentii in 13 (38.2%) patients, Fusobacterium nucleatum subspecies polymorphum in 9 (26.5%) patients, Fusobacterium nucleatum and Fusobacterium species each in 6 (17.6%) patients, Fusobacterium nucleatum subspecies nucleatum in 4 (11.8%) patients, and Fusobacterium nucleatum subspecies animalis in 3 (8.8%) patients. Three patients additionally yielded subgingival isolates of Fusobacterium canifelinum, normally an inhabitant of the oral cavity of dogs and cats. 52 (54.2%) of the fusobacteria clinical isolates revealed MALDI Biotyper log scores of equal to or larger than 1.7, the threshold for reliable taxonomic classification, while in comparison, 44 (45.8%) had log scores less than 1.7, indicating a less reliable species identification. No other microbial species, other than one of the Fusobacterium species, was listed by the MALDI-TOF mass spectrometry analytic software as the most likely organism for the tested clinical isolates. Conclusions: These findings indicate that a variety of Fusobacterium species may be identified with MALDI-TOF mass spectrometry in the subgingival microbiota of chronic periodontitis patients. F. naviforme and F. nucleatum subspecies vincentii were the most frequently isolated subgingival fusobacteria species in the evaluated study patients. Three chronic periodontitis patients also unexpectedly revealed subgingival isolates of the animal species F. canifelinum, which is normally in the oral cavity of dogs and cats. MALDI-TOF mass spectrometry may facilitate rapid identification of cultivable fusobacteria in human subgingival dental plaque biofilms, and enhance understanding of bacterial community structure in periodontal pockets. / Oral Biology

The microbiome has long been an alluring target to study and recent advancements in microbial detection and omics-technologies has further revolutionized our view of how human diseases are impacted by the microbiome. A member of the human microbiome that has garnered such attention is Fusobacterium nucleatum, a Gram-negative, anaerobic bacterium, that normally inhabits the human oral cavity. Interestingly, F. nucleatum is highly invasive into surrounding cells and tissues of the periodontal pocket (below the gymline) and capable of disseminating throughout the entire body. Because of this, F. nucleatum is associated with a wide variety of diseases, most recently and strikingly, colorectal cancer. Despite the pathogenic potential of F. nucleatum, there is limited knowledge about the molecular mechanisms contributing to the invasive nature and virulence of this oral bacterium. This gap in knowledge can be attributed to the absence of genetic tools and resources to investigate and study host-pathogen interactions of Fusobacterium. Progress in dissecting the role of Fusobacterium in disease has been hindered by a lack of fully sequenced and annotated genomes, and the absence of genetic systems to generate target virulence gene deletions to validate mechanisms contributing to host-pathogen interactions. Breakthroughs discussed in this work focus on developing and expanding the genetic toolkits and resources available for studying F. nucleatum interactions in relation to human health and disease. As part of this work, herein, I introduce FusoPortal, an online database of fully sequenced and annotated Fusobacterium genomes, that enabled the bioinformatic annotation and correction of large protein encoding reading frames, that were previously misannotated. This database features a custom basic local alignment search tool (BLAST) server that establishes this resource as a powerful tool for identifying potential virulence factors that contribute to Fusobacterium pathogenesis. Most notably, FusoPortal facilitated my discovery of DNA uptake machinery involved in natural competence and transformation in F. nucleatum. This work is the first to characterize natural competence in a Fusobacterium species, and also enables the expansion of Fusobacterium genetics utilizing the newly found competence mechanism. The findings within this dissertation encompass a paradigm shift in efficient and robust tools to study F. nucleatum biology and pathogenesis. By creating tools for identifying key genes, proteins, and mechanisms involved in Fusobacterium induced or accelerated diseases, there is the potential to accelerate the development of novel therapeutics and vaccines against the emerging 'oncomicrobe' Fusobacterium nucleatum. / Doctor of Philosophy / The trillions of microbes living on or in the human body, collectively called the microbiome, has long been a captivating target to study and understand its role in human health and disease. Recent advances in technology have revolutionized our view of the individual components of the human microbiome, which has led to a renaissance in understanding how specific bacterial species could be used to modulate human health and fight a myriad of diseases. A member of this microbiome that has garnered such attention is Fusobacterium nucleatum, a bacterium that lives in oxygen free pockets along the gumline in the human mouth. A striking feature of F. nucleatum is its ability to invade surrounding tissue, driving bacterial spread throughout the entire body. This bacterium is associated with a wide variety of diseases, most importantly colon cancer. Although F. nucleatum is implicated in these diseases, we still know very little about the mechanisms used by Fusobacterium to promote disease. This roadblock in studying F. nucleatum can largely be attributed to the lack of molecular tools and resources to investigate and study the interactions between the bacteria and its human host. Therefore, research discussed in this work revolved around developing and resources available for studying F. nucleatum interactions in relation to human health and disease. One such resource developed was FusoPortal, an online website with fully sequenced and annotated genomes. This resource was critical in the bioinformatic annotation and correction of large proteins that were previously misannotated. This website features a tool that allows one to search this complete set of genes for a specific sequence establishing this resource as an important tool for identifying key genes and mechanisms that could influence F. nucleatum ability to infect and cause disease. Most notably, FusoPortal provided the means to discover a bacterial system that can import DNA and integrate it into the bacterial genome, a process called natural competence. This work is the first to characterize natural competence in a Fusobacterium species, and has allowed me to utilize the newly found natural competence mechanism to enhance Fusobacterium genetics. In summary, the findings within this dissertation brings about a new horizon for studying F. nucleatum biology, thereby, providing the framework for creating future therapeutic strategies to treat diseases including colorectal cancer.

Fusobacterium nucleatum is a Gram-negative, anaerobic bacterium that is a member of the human oral microbiota. Although it is a normal resident of the mouth, it is associated with a number of human diseases including: sepsis, inflammatory bowel disease (IBD), and colorectal cancer (CRC). Despite the important association of F. nucleatum with human health and disease, remarkably little is known about the molecular mechanisms underlying these infections. This knowledge gap can, in part, be attributed to a lack of molecular tools and experimental workflows. Creating the genetic tools to fill this knowledge gap is an imperative undertaking for the future development of treatments for diseases involving F. nucleatum. Previous work in the field has assigned functions to just a handful of Fusobacterium proteins (Fap2, FadA), and only two of those proteins have a well-defined role in the host-pathogen relationship. This dissertation contains work that lays the molecular and genetic foundation for future studies involving F. nucleatum by creating a unique gene deletion system while simultaneously establishing broadly applicable experimental workflows and molecular tools to study initial bacterial attachment and invasion processes crucial to Fusobacterium virulence. Marker-less gene deletions confirm the importance of Fap2 in host-cell attachment and invasion and suggest a lesser role in invasion for FadA, representing a significant revision to the Fusobacterium-host relationship. Also, our system allows for the overexpression and purification of virulence factors directly from Fusobacterium for the first time. This permits us to study aspects of Fusobacterium protein biology that were previously impossible and will provide further insights into the nature of Fusobacterium virulence. A custom suite of molecular tools was also developed to facilitate recombinant expression of these proteins in general laboratory settings using simple E. coli protein expression systems. We have used these new technologies to express and purify a number of potential Fusobacterium virulence factors as detailed in this dissertation. Also contained in this dissertation is the application of these breakthroughs to probe the function of a novel F. nucleatum outer membrane phospholipase, FplA. Phospholipases are important virulence factors in a number of well-studied human pathogens including Pseudomonas aeruginosa and Legionella pneumophila, where they interfere with host cellular signaling processes to increase intracellular bacterial survival. Our data show that FplA is a Class A1 phospholipase (PLA1) with robust catalytic activity capable of binding to and cleaving a number of lipid types. Additionally, we show that it has the ability to bind to important host signaling lipids including phosphatidylinositol 3, 5-bisphosphate and phosphatidylinositol 3, 4, 5-triphosphate. These data suggest FplA may play a role in manipulating the intracellular processes of host cells. Taken together, work in this dissertation provides tools and experimental frameworks for the future study of F. nucleatum pathogenesis while identifying and initially characterizing a new, potentially significant, virulence factor in FplA. / Doctor of Philosophy

Fusobacterium nucleatum is a Gram-negative bacterium that serves as a bridging organism in polymicrobial biofilms within the oral cavity. Although the bacterium is abundant in healthy gingival tissue, recent studies have found that F. nucleatum is associated with a wide-spectrum of human diseases which include periodontal disease, preterm birth, endocarditis, colorectal cancer, and pancreatic cancer. Previous studies of F. nucleatum virulence have uncovered two surface adhesins, Fap2 and FadA, that interact with the surface of human cells; however, the study of new virulence factors was previously limited as there was no gene deletion system available to functionally analyze F. nucleatum proteins. Interestingly, F. nucleatum has a diverse landscape of structurally unique surface adhesins called Type 5c secreted trimeric autotransporter adhesins (TAAs), which are a family of proteins that are historically known for their contributions to bacterial pathogenesis. This dissertation encompasses the use of recombinant protein expression systems and newly developed gene deletion technology to provide a foundational understanding of the contribution of Type 5c secreted proteins in F. nucleatum pathogenesis. Our results show that the presence of TAAs on the surface of F. nucleatum contribute to the bacterium's ability to bind and invade human cells, establishing the need to characterize other F. nucleatum surface proteins. Additionally, our studies analyzed the proinflammatory landscape induced by F. nucleatum through the identification of specific cytokines that are being secreted during in vitro infections of human cells. Cytokine signaling is a critical aspect of the host cell immune response as it promotes the recruitment of immune cells to the site of infection for efficient clearance of bacterial pathogens. While it has been well established that F. nucleatum modulates the secretion of IL-8, our studies identified that the bacterium also promotes the secretion of CXCL1, which is an important signaling protein that promotes tumor metastases. Overall, the work provided in this dissertation has delivered the initial characterization of TAAs in F. nucleatum virulence, a framework for future studies of Type 5c secreted proteins in Fusobacterium pathogenesis, and the role of Fap2 and FadA in promoting pro-inflammatory and pro-metastatic signaling from colorectal cancer cells. / Master of Science in Life Sciences / Fusobacterium nucleatum is a Gram-negative bacterium that serves as a bridging organism in polymicrobial biofilms within the oral cavity. Although the bacterium is abundant in healthy gingival tissue, recent studies have found that F. nucleatum is associated with a wide-spectrum of human diseases which include periodontal disease, preterm birth, endocarditis, colorectal cancer, and pancreatic cancer. Previous studies of F. nucleatum virulence have uncovered two surface adhesins, Fap2 and FadA, that interact with the surface of human cells; however, the study of new virulence factors was previously limited as there was no gene deletion system available to functionally analyze F. nucleatum proteins. Interestingly, F. nucleatum has a diverse landscape of structurally unique surface adhesins called Type 5c secreted trimeric autotransporter adhesins (TAAs), which are a family of proteins that are historically known for their contributions to bacterial pathogenesis. This dissertation encompasses the use of recombinant protein expression systems and newly developed gene deletion technology to provide a foundational understanding of the contribution of Type 5c secreted proteins in F. nucleatum pathogenesis. Our results show that the presence of TAAs on the surface of F. nucleatum contribute to the bacterium’s ability to bind and invade human cells, establishing the need to characterize other F. nucleatum surface proteins. Additionally, our studies analyzed the proinflammatory landscape induced by F. nucleatum through the identification of specific cytokines that are being secreted during in vitro infections of human cells. Cytokine signaling is a critical aspect of the host cell immune response as it promotes the recruitment of immune cells to the site of infection for efficient clearance of bacterial pathogens. While it has been well established that F. nucleatum modulates the secretion of IL-8, our studies identified that the bacterium also promotes the secretion of CXCL1, which is an important signaling protein that promotes tumor metastases. Overall, the work provided in this dissertation has delivered the initial characterization of TAAs in F. nucleatum virulence, a framework for future studies of Type 5c secreted proteins in Fusobacterium pathogenesis, and the role of Fap2 and FadA in promoting pro-inflammatory and pro-metastatic signaling from colorectal cancer cells

La maladie parodontale est la maladie infectieuse la plus répandue chez les animaux de compagnie. À partir de l'âge de deux ans, près de 80 % des chiens et 70 % des chats en sont atteints. Les bactéries contenues dans la plaque dentaire sont la cause première du développement de la maladie parodontale. De plus, la réponse immunitaire non contrôlée de l'hôte peut mener à la destruction du parodonte. Le but du projet de recherche est de tester l'efficacité de cinq produits naturels contre quatre espèces de Porphyromonas et une espèce de Fusobacterium de source animale. L'objectif est d'identifier un produit naturel qui permettrait de prévenir ou traiter la maladie parodontale et l'halitose chez le chien et le chat. Le projet a permis d'évaluer les effets antibactériens et anti-adhérence ainsi que les effets sur la production de composés sulfurés volatils des bactéries à l'étude. Ainsi, il pourrait s'avérer possible d'améliorer la santé et l'espérance de vie des animaux à l'aide d'une alternative plus facile et moins stressante que le brossage de dents. Ce projet a démontré que la plupart des produits naturels à l'étude possèdent des propriétés antibactériennes, anti-adhérence et diminuent également la production de composés sulfurés volatils. En conclusion, les résultats obtenus dans cette étude ont apporté des évidences supportant le potentiel de certains produits naturels d'être utilisés dans la prévention ou le traitement de la maladie parodontale et de l'halitose chez le chat et le chien.

Fusobacterium nucleatum is an anaerobic, Gram-negative, oral bacterium that disseminates from the mouth, and contributes to preterm birth, tissue infections, and acceleration of multiple cancers including colorectal and pancreatic. It is well-established that most Fusobacterium species exhibit genetic recalcitrance, which has led to hindrance in the understanding of their biology and molecular pathogenesis. Though the association of Fusobacterium in diseases is well-established, the majority of our experimental work stems from the strain F. nucleatum ATCC 23726 because it is genetically tractable. Here, in this dissertation, we show that we are able to enhance our existing molecular tools for genome editing to introduce the first mutants in a clinically relevant strain, F. nucleatum ATCC 25586, a feat that was never accomplished in decades of trying. Furthermore, we created a deletion library of genes predicted to be involved in host cellular invasion and survival. In this work, we identified a novel small adhesin, FadA2, that played a significant role in the invasive ability of F. nucleatum ATCC 25586 to colorectal cancer cells. This dissertation also sheds the first insight into the roles of the type 5a autotransporters. Using a deletion library of genes encoding for the type 5a autotransporter proteins in F. nucleatum ATCC 23726, we systemically characterized altogether 12 type 5a proteins with a focus on the invasion of colorectal cancer cells. Most notably, we found that a wide assortment of type 5a proteins contributing to binding and invasion of F. nucleatum to HCT116 cancer cells. Furthermore, we identified that RadD was not directly involved in inducing secretions of the cytokines IL-8 and CXCL1 while confirmed the specific association of Fap2 in bacterial-induced cytokine secretion. Thus, our findings provided the first comparative and functional analysis of Fusobacterium type 5a autotransporter proteins in colorectal cancer cells which will be crucial to the understanding of Fusobacterium involvement in cancer progression. Finally, this dissertation reported on the first ever observation on the survival strategy of Fusobacterium inside the host cells. We uncovered a novel protein that contributed to enhanced survival of Fusobacterium residing in colorectal cancer cells. This work undoubtedly helps expand the current Fusobacterium genetic toolkit to study proteins and mechanisms relevant to Fusobacterium-accelerated diseases. By identifying and characterizing novel virulence strategies that Fusobacterium can take advantage of, we can increase our comprehension on this opportunistic microbe while devising innovative therapeutic treatments. / Doctor of Philosophy / Fusobacterium, a member of the microbial community in our mouth, has been a captivating study target due to its association with human health and diseases. By nature, Fusobacterium lives in oxygen-free pockets between our teeth and gumline in which this organism has been correlated with a multitude of complications and diseases including periodontitis, inflammatory bowel disease, preterm birth, and most importantly colorectal cancer. Though the connection to human health is established, we still have to learn more about the mechanisms utilized by Fusobacterium to exacerbate diseases. This challenge is mainly hindered by the lack of efficient tools and resources to systematically investigate the relationship between the bacterium and its human host. Therefore, the work in this dissertation focuses on expanding the existing molecular toolkit to study clinically relevant Fusobacterium strain, which provides the power and convenience to discover novel mechanisms that Fusobacterium can take advantage of to be a successful pathogen. Accordingly, we first enhanced our ability to work with a wider range of Fusobacterium species. We successfully introduced exogenous genetic materials into a clinical strain of Fusobacterium, Fusobacterium nucleatum ATCC 25586. This breakthrough was built on the success of our current toolkit to make genetic modifications to a sister strain, Fusobacterium nucleatum ATCC 23726. With this newfound capacity to modify F. nucleatum ATCC 25586, we have described the importance of a novel protein aiding in the invasion of Fusobacterium to colorectal cancer. Furthermore, we have determined that certain proteins within the fusobacterial type 5a protein family can play a key role in governing binding and invasion of colorectal cancer cells in this study. Concurrently, for the first time, we provided the snapshot of a small protein and its role in fusobacterial long-term survival inside its targeted host cells. Altogether, the findings in this dissertation will bring forth an innovative framework to better the comprehension of current Fusobacterium-induced disease implications, while exploring alternative treatments for enhanced patient health.

Dans ce projet de recherche, les propriétés antibactériennes et le potentiel anti-destruction tissulaire des polyphénols du thé vert, plus particulièrement l’épigallocatéchine-gallate (EGCG), ont été évalués. Dans un premier temps, l’effet antibactérien d’un extrait de thé vert et de l’EGCG a été déterminé sur trois bactéries parodontopathogènes d’importance, soit Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans et Fusobacterium nucleatum. Les concentrations minimales inhibitrices se sont avérées être entre 1000 et 62,5 µg/ml. De plus, des effets synergiques et additifs des polyphénols du thé vert ont été observés lorsqu’utilisés en association avec le métronidazole ou la tétracycline, des antibiotiques couramment utilisés en thérapie parodontale. Dans un deuxième temps, un modèle de co-culture constitué de fibroblastes gingivaux intégrés dans un gel de collagène et recouvert de macrophages a été utilisé pour évaluer la capacité des polyphénols du thé vert à inhiber la sécrétion de métalloprotéinases matricielles (MMPs), notamment les MMP-3, MMP-8 et MMP-9. Il a été démontré que les polyphénols du thé vert atténuent la sécrétion des MMPs par le modèle de co-culture. En conclusion, les résultats de cette étude ont apporté des évidences supportant le potentiel des polyphénols du thé vert en vue d’une utilisation préventive et thérapeutique pour le contrôle des maladies parodontales.

La parodontite est une infection polymicrobienne qui entraîne une inflammation du parodonte et provoque une destruction irréversible des tissus de soutien de la dent. Le but de cette étude était d'évaluer les concentrations des cytokines IL-18, IL-1β, IL-10, TNFα ainsi que celles des récepteurs Toll de type 2 (Toll-like receptor 2, TLR2) et 4 (TLR4) solubles dans le fluide créviculaire gingivale (FCG) et la salive d'une cohorte de patients atteints de parodontite. Ces concentrations ont été corrélées avec les paramètres cliniques de la parodontite et la quantité des bactéries Porphyromonas gingivalis, Treponema denticola et Fusobacterium nucleatum retrouvées chez les participants à l'étude. Les résultats obtenus ont révélé que les taux d'IL-18, d'IL-1β et de TLR4 dans le FCG sont plus élevés chez les sujets atteints de parodontite par rapport à ceux exempts de la maladie et que la sévérité de la maladie parodontale est associée à des concentrations élevées d'IL-1β et de TLR4 chez les sujets malades.

In der vorliegenden Arbeit sollten die in der veterinärmedizinischen Literatur bisher diskutierten Ursachen für LJD bei Makropoden hinsichtlich ihrer tatsächlichen Bedeutung abgeklärt und die Eignung einer formalininaktivierten, bestandsspezifischen Adsorbatvakzine zur Prophylaxe von LJD getestet werden. Da LJD eine parodontale Erkrankung darstellt, wurden auch die für Entstehung einer humanen Parodontitis prädisponierenden Faktoren mit in die Untersuchung einbezogen. Es wurden Tupferproben zur bakteriologischen Untersuchung von insgesamt 15 gesunden und 11 an LJD erkrankten Kängurus entnommen. Dabei konnten gramnegative Anaerobier bei allen Tieren isoliert werden. Fusobacterium nucleatum wurde in 82% der von an LJD erkrankten und nur in 33% der von gesunden Tieren entnommenen Tupferproben nachgewiesen, womit sich ein signifikanter Zusammenhang (P < 0,05) zwischen diesem Erreger und LJD ergab. Weitere überwiegend bei erkrankten Makropoden nachgewiesene Anaerobier stellten Prevotella oris/oralis (bei 73% der LJD-Fälle und bei 40% der gesunden Tiere) sowie Capnocytophaga spp. (45% vs. 13%) dar. Bacteroides spp. und Porphyromonas gingivalis wurden – wenn auch nur mit 3 bzw. 2 Nachweisen – ausschließlich bei kranken Tieren isoliert. Fusobacterium necrophorum wurde jeweils in 27% der Kängurus gefunden und spielte damit in dieser Studie keine Rolle für die Entstehung von LJD. In Übereinstimmung mit der Literatur konnten Moraxella spp. ausschließlich bei gesunden Makropoden isoliert werden. Vertreter dieser Gattung gehören damit offensichtlich zur normalen Maulflora der Kängurus. Für die Zoos in Halle und Leipzig wurde eine formalininaktivierte, bestandsspezifische Adsorbatvakzine gegen die bei einem an LJD erkrankten Känguru des jeweiligen Bestandes isolierten gramnegativen Anaerobier hergestellt. 7 Tiere (2 Rote Riesenkängurus, 5 Bennettwallabies) des Leipziger Zoos und 6 Bennettkängurus des Zoos in Halle wurden geimpft, wobei Auffrischungsimpfungen nach 4 bzw. 8 Wochen und nach 6 bzw. 12 Monaten erfolgten. Die spezifischen AK gegen das Prüfantigen Fusobacterium necrophorum wurden im SLA bestimmt. Es konnte keine Erhöhung der AK-Titer induziert werden und auch die Todesrate infolge von LJD senkte sich während des Untersuchungszeitraumes von 42 Monaten in den beiden Zoos nicht. Die höchsten AK-Level (1:512 bis 1:2048) ließen sich im Serum von natürlich infizierten und letztendlich tödlich erkrankten Bennettwallabies des Zoos in Hoyerswerda feststellen. Der Nachweis von AK-Titern im Serum von nicht geimpften Jungtieren lässt vermuten, dass AK via Kolostrum oder Dottersackplazenta auf die Jungtiere übertragen werden. Die Untersuchungen hinsichtlich der Fütterung zeigten, dass im Zoo Leipzig eine azidotische Stoffwechsellage induziert wurde, was sich bei den Leipziger Bennettkängurus in einem mit 7,53 signifikant niedrigeren Vormagen-pH-Wert im Vergleich zu den Hallenser und Auer Tieren (8,25 und 8,38) offenbarte. Dies schlug sich auch in erhöhten K-, Cholesterol- und &#61537;-Amylasewerten im Serum der Leipziger Wallabies nieder, womit gezeigt werden konnte, dass sich diese Parameter offenbar auch bei Makropoden zur Diagnostik einer chronischen Azidose eignen. Die Versorgung der Bennettkängurus in Magdeburg und Halle mit Ca und P war zwar nicht ausreichend, spiegelte sich aber nicht in veränderten Blutwerten dieser Mengenelemente wider. Die Aktivität der AP nimmt mit zunehmenden Alter ähnlich wie bei anderen Tierarten ab. Ihre negative Korrelation mit dem Alter der Tiere war dabei hochsignifikant (P < 0,001, r = 0,77 bzw. 0,62). Beim direkten Vergleich gesunder mit an LJD erkrankten Tieren konnte weder eine Störung im Ca/P-Stoffwechsel noch eine Azidose in Verbindung zu LJD gebracht werden. In allen Zoos erfolgte eine Überversorgung mit Vitamin A, wobei die Bedarfswerte für Schaflämmer um das 3,5fache bis 41fache übertroffen wurden. Den Bedarfswerten am nächsten lagen die Versorgungswerte der Bennettkängurus vom TP Aue und der Östlichen Grauen Riesenkängurus vom Zoo Magdeburg, beides Bestände ohne LJD. Die ermittelten Retinolplasmakonzentrationen standen in keiner Beziehung zu den Vitamin-A-Gehalten im Futter, was darauf hindeutet, dass sich Retinolbestimmungen im Blutplasma ebenso wie bei anderen Tierarten nur in extremsten defizitären Situationen zur Einschätzung des Vitamin-A-Status eignen. Ob eine Hypervitaminose A für die Entstehung von LJD tatsächlich eine Rolle spielt, muss in zukünftigen Arbeiten unter Einbeziehung von Retinolesterbestimmungen in der Leber abgeklärt werden. Die Glukosewerte lagen mit 8,57 mmol/l (M. rufus) bzw. 6,51 mmol/l (M. rufogriseus) über den bisher bekannten Werten aus der Literatur. Da die Werte bei an LJD erkrankten Kängurus niedriger waren als bei gesunden Tieren, kann ein Diabetes mellitus als Ursache für LJD ausgeschlossen werden. Weder die Durchsicht von 144 Sektionsprotokollen noch die Bestimmung der Kreatinin- und Harnstoffkonzentration im Serum von an LJD erkrankten Tieren ließen einen Zusammenhang zwischen Erkrankungen der Nieren und LJD erkennen. 30 Tiere verendeten an LJD, wovon 20% auch an den Nieren erkrankt waren. Allerdings wiesen auch 16,7% der anderweitig gestorbenen Kängurus eine Nierenerkrankung auf. Die Serumkonzentrationen von Harnstoff bzw. Kreatinin der an LJD erkrankten Makropoden unterschieden sich nicht von den für die gesunden Roten Riesenkängurus (7,40 mmol bzw. 114 mmol/l) und Bennettwallabies (7,81 mmol/l bzw. 86 mmol/l) ermittelten Werten. Insgesamt 184 Sera von 107 Kängurus wurden auf AK gegen MaHV-1 und MaHV-2 mittels Neutralisationtest geprüft. Während 94,4% bzw. 97,2% der Roten Riesenkängurus serologisch positiv für MaHV-1 bzw. MaHV-2 waren, reagierten von den 71 überprüften Bennettkängurus nur 4 bzw. 3 Tiere positiv. Unter den Wallabies befanden sich auch 21 an LJD erkrankte Tiere, wovon lediglich 2 Tiere gegen MaHV-1 und 1 Tier gegen MaHV-2 eine Serokonversion zeigten. Die AK-Titer der Roten Riesenkängurus ließen keine Unterschiede zwischen gesunden und an LJD leidenden Tieren zu und die entnommenen Serumpaarproben von 5 zum Zeitpunkt der Blutentnahme an LJD leidenden Riesenkängurus zeigten kein einheitliches Verhalten im Sinne einer Serokonversion. Somit ließ sich der Verdacht, dass die Reaktivierung latenter Herpesinfektionen die Ursache für LJD sein könnte, nicht bestätigen. Im Ergebnis der vorliegenden Studie und im Zusammenhang mit den Angaben aus der Literatur stellt sich LJD primär als eine Infektion mit gramnegativen Anaerobiern dar, wovon Fusobacterium nucleatum, Bacteroides spp., Prophyromonas gingivalis und Fusobacterium necrophorum, Biovar A die größte Bedeutung haben dürften. Den Abschluss der Arbeit bilden Empfehlungen für die Haltung von Kängurus in zoologischen Einrichtungen und für die Therapie von LJD. Im Anhang finden sich Röntgenaufnahmen und Photographien von erkrankten und gesunden Makropoden. / The aim of this thesis was the investigation of the aetiology of Lumpy Jaw Disease (LJD) in macropods concentrating specifically on the causes of the diseases in current veterinary medicine literature and to evaluate the use of a group-specific Al(OH)3-adjuvanted, formalin-inactivated whole-cell vaccine for the control of LJD in kangaroos kept in zoos. LJD is regarded as periodontal disease, therefore the risk factors for the development of human periodontitis were also included in this study. The oral flora from 15 healthy macropods and 11 animals suffering from LJD was isolated. At least one anaerobic gram-negative bacterial species was found in swabs of each macropod. The occurrence of Fusobacterium nucleatum was associated with LJD (P < 0.05) by detecting this bacterium in 82% of the kangaroos suffering from LJD compared to only in 33% of the healthy animals. Prevotella oris/oralis and Capnocytophaga spp. were also predominantly found in diseased animals in comparison with healthy macropods (73% vs. 40% and 45% vs. 13% respectively). Bacteroides spp. and Porphyromonas gingivalis were isolated in only 3 and 2 kangaroos suffering from LJD, respectively. Contrary to previously published studies about LJD Fusobacterium necrophorum was not associated with LJD, as this anaerobe was detected in only 27% of the diseased as well as healthy macropods. Moraxella spp. seem to be a part of the normal oral flora of macropods and was found exclusively in healthy animals. 11 Red-necked Wallabies (Macropus rufogriseus) and 2 Red Kangaroos (Macropus rufus) were immunized with a group-specific Al(OH)3-adjuvanted, formalin-inactivated whole-cell vaccine containing previously in a kangaroo suffering from LJD isolated gramnegative anaerobs. The kangaroos were re-vaccinated after 1, 2, 6 and 12 months. Blood was collected from each animal at the same time. Antibodies were titrated against Fusobacterium necrophorum in an agglutination assay. The vaccine failed to induce increased levels of antibodies as well as to protect wallabies and kangaroos against LJD. As the highest antibody titres were detected in most severely diseased wallabies kept in the Hoyerswerda zoo, the protective role of the humoral immune response in LJD seems to be doubtful. The finding of detectable levels of antibodies in unvaccinated joeys supports the theory, that there is a transmission of antibodies from the mother to the offspring via colostrum or yolk-sac placenta. The diet of the Red-necked Wallabies in one zoo has induced an acidosis: The pH of the forestomach fluid collected by probang was lower in the animals of this zoo (pH = 7.53) than in the wallabies of two other zoos (pH = 8.25 and 8.38, respectively). Potassium, cholesterol and &#61537;-amylase were also higher in the blood of the animals of this zoo in comparison to the wallabies of the two other ones, hence these blood values seem to be helpful for the diagnosis of chronic acidosis in macropods. There was a calcium and phosphor deficiency in the nutrition of the wallabies in two zoos, but the blood concentration of both of these minerals was not changed. The activity of the ALP correlated negative with the age of the Bennett`s Wallabies (P < 0.001, r = -.77 and r = -.62 respectively, depending on the instruments). All of the above mentioned blood values showed no differences between healthy and diseased animals and could so far not support the assumption, that an imbalance in Ca and P metabolism or an acidosis are important factors for LJD. The macropods of all investigated zoos were fed on a diet rich in vitamin A ranging from the 3.5 to the 41fold requirement for lambs. The vitamin A content of the diets for the 2 collections without a history of LJD was the lowest in this study. These results raised the point, that a hypervitaminosis A could be a more predisposing factor for LJD than a vitamin A deficiency. Due to the fact the plasma retinol concentration was independent from the vitamin A content of the diet and so not helpful in diagnosis of a vitamin A deficiency or toxicity, further investigations regarding the role of vitamin A in the aetiopathogenesis of LJD should include measurements of the liver tissue content of retinol esters. The glucose plasma concentration of the healthy Red Kangaroos (8.57 mmol/l) as well as the Red-necked Wallabies (6.51 mmol/l) was higher than previously published values for macropods, but also higher than the results of the diseased animals in this study. Therefore diabetes mellitus can be ruled out as an underlying factor for LJD. The analysis of 144 pathological records showed, that 30 animals died because of LJD, 20% of them and 16.7% of the other 114 macropods had a concurrent kidney disease. The urea and creatinin concentration in serum samples of healthy animals was not higher than the values of diseased animals. In conclusion, these results suggest kidney diseases are not important for the development of LJD. Altogether 184 sera collected from 107 kangaroos were tested for antibodies against MaHV-1 and MaHV-2 using a neutralisation assay. The prevalence of the MaHV-1- as well as MaHV-2-antibodies was high among the Red Kangaroos (94.4% and 97.2% respectively), but low among the Red-necked Wallabies (5.6% and 4.2% respectively). Seroconversion for MaHV-1 was seen in 2 out of 21 wallabies suffering from LJD, only 1 of these animals also had antibodies against MaHV-2. The antibody-titres against both of the macropodid herpes viruses also did not differ between Red Kangaroos with and without LJD, therefore a reactivation of a latent herpesvirus infection does not appear to be causative for LJD. In summary, considering the results of this study and previously published literature LJD is an infectious disease caused by gramnegative anaerobic bacteria with Fusobacterium nucleatum, Bacteroides spp., Porphyromonas gingivalis and Fusobacterium necrophorum subsp. necrophorum being of most significance. Recommendations concerning the keeping of kangaroos in captivity and the management of LJD are listed in the conclusion of this thesis. Some radiographs and photos of diseased and healthy kangaroos are attached.

In der vorliegenden Arbeit sollten die in der veterinärmedizinischen Literatur bisher diskutierten Ursachen für LJD bei Makropoden hinsichtlich ihrer tatsächlichen Bedeutung abgeklärt und die Eignung einer formalininaktivierten, bestandsspezifischen Adsorbatvakzine zur Prophylaxe von LJD getestet werden. Da LJD eine parodontale Erkrankung darstellt, wurden auch die für Entstehung einer humanen Parodontitis prädisponierenden Faktoren mit in die Untersuchung einbezogen. Es wurden Tupferproben zur bakteriologischen Untersuchung von insgesamt 15 gesunden und 11 an LJD erkrankten Kängurus entnommen. Dabei konnten gramnegative Anaerobier bei allen Tieren isoliert werden. Fusobacterium nucleatum wurde in 82% der von an LJD erkrankten und nur in 33% der von gesunden Tieren entnommenen Tupferproben nachgewiesen, womit sich ein signifikanter Zusammenhang (P < 0,05) zwischen diesem Erreger und LJD ergab. Weitere überwiegend bei erkrankten Makropoden nachgewiesene Anaerobier stellten Prevotella oris/oralis (bei 73% der LJD-Fälle und bei 40% der gesunden Tiere) sowie Capnocytophaga spp. (45% vs. 13%) dar. Bacteroides spp. und Porphyromonas gingivalis wurden – wenn auch nur mit 3 bzw. 2 Nachweisen – ausschließlich bei kranken Tieren isoliert. Fusobacterium necrophorum wurde jeweils in 27% der Kängurus gefunden und spielte damit in dieser Studie keine Rolle für die Entstehung von LJD. In Übereinstimmung mit der Literatur konnten Moraxella spp. ausschließlich bei gesunden Makropoden isoliert werden. Vertreter dieser Gattung gehören damit offensichtlich zur normalen Maulflora der Kängurus. Für die Zoos in Halle und Leipzig wurde eine formalininaktivierte, bestandsspezifische Adsorbatvakzine gegen die bei einem an LJD erkrankten Känguru des jeweiligen Bestandes isolierten gramnegativen Anaerobier hergestellt. 7 Tiere (2 Rote Riesenkängurus, 5 Bennettwallabies) des Leipziger Zoos und 6 Bennettkängurus des Zoos in Halle wurden geimpft, wobei Auffrischungsimpfungen nach 4 bzw. 8 Wochen und nach 6 bzw. 12 Monaten erfolgten. Die spezifischen AK gegen das Prüfantigen Fusobacterium necrophorum wurden im SLA bestimmt. Es konnte keine Erhöhung der AK-Titer induziert werden und auch die Todesrate infolge von LJD senkte sich während des Untersuchungszeitraumes von 42 Monaten in den beiden Zoos nicht. Die höchsten AK-Level (1:512 bis 1:2048) ließen sich im Serum von natürlich infizierten und letztendlich tödlich erkrankten Bennettwallabies des Zoos in Hoyerswerda feststellen. Der Nachweis von AK-Titern im Serum von nicht geimpften Jungtieren lässt vermuten, dass AK via Kolostrum oder Dottersackplazenta auf die Jungtiere übertragen werden. Die Untersuchungen hinsichtlich der Fütterung zeigten, dass im Zoo Leipzig eine azidotische Stoffwechsellage induziert wurde, was sich bei den Leipziger Bennettkängurus in einem mit 7,53 signifikant niedrigeren Vormagen-pH-Wert im Vergleich zu den Hallenser und Auer Tieren (8,25 und 8,38) offenbarte. Dies schlug sich auch in erhöhten K-, Cholesterol- und &#61537;-Amylasewerten im Serum der Leipziger Wallabies nieder, womit gezeigt werden konnte, dass sich diese Parameter offenbar auch bei Makropoden zur Diagnostik einer chronischen Azidose eignen. Die Versorgung der Bennettkängurus in Magdeburg und Halle mit Ca und P war zwar nicht ausreichend, spiegelte sich aber nicht in veränderten Blutwerten dieser Mengenelemente wider. Die Aktivität der AP nimmt mit zunehmenden Alter ähnlich wie bei anderen Tierarten ab. Ihre negative Korrelation mit dem Alter der Tiere war dabei hochsignifikant (P < 0,001, r = 0,77 bzw. 0,62). Beim direkten Vergleich gesunder mit an LJD erkrankten Tieren konnte weder eine Störung im Ca/P-Stoffwechsel noch eine Azidose in Verbindung zu LJD gebracht werden. In allen Zoos erfolgte eine Überversorgung mit Vitamin A, wobei die Bedarfswerte für Schaflämmer um das 3,5fache bis 41fache übertroffen wurden. Den Bedarfswerten am nächsten lagen die Versorgungswerte der Bennettkängurus vom TP Aue und der Östlichen Grauen Riesenkängurus vom Zoo Magdeburg, beides Bestände ohne LJD. Die ermittelten Retinolplasmakonzentrationen standen in keiner Beziehung zu den Vitamin-A-Gehalten im Futter, was darauf hindeutet, dass sich Retinolbestimmungen im Blutplasma ebenso wie bei anderen Tierarten nur in extremsten defizitären Situationen zur Einschätzung des Vitamin-A-Status eignen. Ob eine Hypervitaminose A für die Entstehung von LJD tatsächlich eine Rolle spielt, muss in zukünftigen Arbeiten unter Einbeziehung von Retinolesterbestimmungen in der Leber abgeklärt werden. Die Glukosewerte lagen mit 8,57 mmol/l (M. rufus) bzw. 6,51 mmol/l (M. rufogriseus) über den bisher bekannten Werten aus der Literatur. Da die Werte bei an LJD erkrankten Kängurus niedriger waren als bei gesunden Tieren, kann ein Diabetes mellitus als Ursache für LJD ausgeschlossen werden. Weder die Durchsicht von 144 Sektionsprotokollen noch die Bestimmung der Kreatinin- und Harnstoffkonzentration im Serum von an LJD erkrankten Tieren ließen einen Zusammenhang zwischen Erkrankungen der Nieren und LJD erkennen. 30 Tiere verendeten an LJD, wovon 20% auch an den Nieren erkrankt waren. Allerdings wiesen auch 16,7% der anderweitig gestorbenen Kängurus eine Nierenerkrankung auf. Die Serumkonzentrationen von Harnstoff bzw. Kreatinin der an LJD erkrankten Makropoden unterschieden sich nicht von den für die gesunden Roten Riesenkängurus (7,40 mmol bzw. 114 mmol/l) und Bennettwallabies (7,81 mmol/l bzw. 86 mmol/l) ermittelten Werten. Insgesamt 184 Sera von 107 Kängurus wurden auf AK gegen MaHV-1 und MaHV-2 mittels Neutralisationtest geprüft. Während 94,4% bzw. 97,2% der Roten Riesenkängurus serologisch positiv für MaHV-1 bzw. MaHV-2 waren, reagierten von den 71 überprüften Bennettkängurus nur 4 bzw. 3 Tiere positiv. Unter den Wallabies befanden sich auch 21 an LJD erkrankte Tiere, wovon lediglich 2 Tiere gegen MaHV-1 und 1 Tier gegen MaHV-2 eine Serokonversion zeigten. Die AK-Titer der Roten Riesenkängurus ließen keine Unterschiede zwischen gesunden und an LJD leidenden Tieren zu und die entnommenen Serumpaarproben von 5 zum Zeitpunkt der Blutentnahme an LJD leidenden Riesenkängurus zeigten kein einheitliches Verhalten im Sinne einer Serokonversion. Somit ließ sich der Verdacht, dass die Reaktivierung latenter Herpesinfektionen die Ursache für LJD sein könnte, nicht bestätigen. Im Ergebnis der vorliegenden Studie und im Zusammenhang mit den Angaben aus der Literatur stellt sich LJD primär als eine Infektion mit gramnegativen Anaerobiern dar, wovon Fusobacterium nucleatum, Bacteroides spp., Prophyromonas gingivalis und Fusobacterium necrophorum, Biovar A die größte Bedeutung haben dürften. Den Abschluss der Arbeit bilden Empfehlungen für die Haltung von Kängurus in zoologischen Einrichtungen und für die Therapie von LJD. Im Anhang finden sich Röntgenaufnahmen und Photographien von erkrankten und gesunden Makropoden. / The aim of this thesis was the investigation of the aetiology of Lumpy Jaw Disease (LJD) in macropods concentrating specifically on the causes of the diseases in current veterinary medicine literature and to evaluate the use of a group-specific Al(OH)3-adjuvanted, formalin-inactivated whole-cell vaccine for the control of LJD in kangaroos kept in zoos. LJD is regarded as periodontal disease, therefore the risk factors for the development of human periodontitis were also included in this study. The oral flora from 15 healthy macropods and 11 animals suffering from LJD was isolated. At least one anaerobic gram-negative bacterial species was found in swabs of each macropod. The occurrence of Fusobacterium nucleatum was associated with LJD (P < 0.05) by detecting this bacterium in 82% of the kangaroos suffering from LJD compared to only in 33% of the healthy animals. Prevotella oris/oralis and Capnocytophaga spp. were also predominantly found in diseased animals in comparison with healthy macropods (73% vs. 40% and 45% vs. 13% respectively). Bacteroides spp. and Porphyromonas gingivalis were isolated in only 3 and 2 kangaroos suffering from LJD, respectively. Contrary to previously published studies about LJD Fusobacterium necrophorum was not associated with LJD, as this anaerobe was detected in only 27% of the diseased as well as healthy macropods. Moraxella spp. seem to be a part of the normal oral flora of macropods and was found exclusively in healthy animals. 11 Red-necked Wallabies (Macropus rufogriseus) and 2 Red Kangaroos (Macropus rufus) were immunized with a group-specific Al(OH)3-adjuvanted, formalin-inactivated whole-cell vaccine containing previously in a kangaroo suffering from LJD isolated gramnegative anaerobs. The kangaroos were re-vaccinated after 1, 2, 6 and 12 months. Blood was collected from each animal at the same time. Antibodies were titrated against Fusobacterium necrophorum in an agglutination assay. The vaccine failed to induce increased levels of antibodies as well as to protect wallabies and kangaroos against LJD. As the highest antibody titres were detected in most severely diseased wallabies kept in the Hoyerswerda zoo, the protective role of the humoral immune response in LJD seems to be doubtful. The finding of detectable levels of antibodies in unvaccinated joeys supports the theory, that there is a transmission of antibodies from the mother to the offspring via colostrum or yolk-sac placenta. The diet of the Red-necked Wallabies in one zoo has induced an acidosis: The pH of the forestomach fluid collected by probang was lower in the animals of this zoo (pH = 7.53) than in the wallabies of two other zoos (pH = 8.25 and 8.38, respectively). Potassium, cholesterol and &#61537;-amylase were also higher in the blood of the animals of this zoo in comparison to the wallabies of the two other ones, hence these blood values seem to be helpful for the diagnosis of chronic acidosis in macropods. There was a calcium and phosphor deficiency in the nutrition of the wallabies in two zoos, but the blood concentration of both of these minerals was not changed. The activity of the ALP correlated negative with the age of the Bennett`s Wallabies (P < 0.001, r = -.77 and r = -.62 respectively, depending on the instruments). All of the above mentioned blood values showed no differences between healthy and diseased animals and could so far not support the assumption, that an imbalance in Ca and P metabolism or an acidosis are important factors for LJD. The macropods of all investigated zoos were fed on a diet rich in vitamin A ranging from the 3.5 to the 41fold requirement for lambs. The vitamin A content of the diets for the 2 collections without a history of LJD was the lowest in this study. These results raised the point, that a hypervitaminosis A could be a more predisposing factor for LJD than a vitamin A deficiency. Due to the fact the plasma retinol concentration was independent from the vitamin A content of the diet and so not helpful in diagnosis of a vitamin A deficiency or toxicity, further investigations regarding the role of vitamin A in the aetiopathogenesis of LJD should include measurements of the liver tissue content of retinol esters. The glucose plasma concentration of the healthy Red Kangaroos (8.57 mmol/l) as well as the Red-necked Wallabies (6.51 mmol/l) was higher than previously published values for macropods, but also higher than the results of the diseased animals in this study. Therefore diabetes mellitus can be ruled out as an underlying factor for LJD. The analysis of 144 pathological records showed, that 30 animals died because of LJD, 20% of them and 16.7% of the other 114 macropods had a concurrent kidney disease. The urea and creatinin concentration in serum samples of healthy animals was not higher than the values of diseased animals. In conclusion, these results suggest kidney diseases are not important for the development of LJD. Altogether 184 sera collected from 107 kangaroos were tested for antibodies against MaHV-1 and MaHV-2 using a neutralisation assay. The prevalence of the MaHV-1- as well as MaHV-2-antibodies was high among the Red Kangaroos (94.4% and 97.2% respectively), but low among the Red-necked Wallabies (5.6% and 4.2% respectively). Seroconversion for MaHV-1 was seen in 2 out of 21 wallabies suffering from LJD, only 1 of these animals also had antibodies against MaHV-2. The antibody-titres against both of the macropodid herpes viruses also did not differ between Red Kangaroos with and without LJD, therefore a reactivation of a latent herpesvirus infection does not appear to be causative for LJD. In summary, considering the results of this study and previously published literature LJD is an infectious disease caused by gramnegative anaerobic bacteria with Fusobacterium nucleatum, Bacteroides spp., Porphyromonas gingivalis and Fusobacterium necrophorum subsp. necrophorum being of most significance. Recommendations concerning the keeping of kangaroos in captivity and the management of LJD are listed in the conclusion of this thesis. Some radiographs and photos of diseased and healthy kangaroos are attached.