Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 93 pages. Includes vita. Includes bibliographical references.

Tularemia is a vector-borne disease of global concern with diverse regional foci. Arkansas is an endemic state with differences in case distribution and land suitability supporting host and vector sustainment. The aim of this study was to conduct a geospatial and spatiotemporal assessment of factors associated with case distribution and timeliness and completeness of public reporting. Guided with direction from spatial epidemiology and nidality, referring to the association of ecology, climate, and proximity of disease, analysis included secondary data collected from the Arkansas Department of Health between 1995 and 2018. Using Poisson-based software, 2 clusters were found: a high-risk cluster encompassing 23% of the total population within 24 counties spanning an 8-year period (RR = 4.98, p < 0.05), and a low risk cluster that included 25% of the population within 28 counties during a 12-year period (RR 0.14, p < 0.05). Analysis of ecological data revealed associations between annual precipitation within the high-risk cluster and total number of cases (AUC = 0.716 and AUC = 0.726, respectively) with trends toward higher incidence rates in suitable land cover and moderate to high elevation using maximum entropy software. Analysis of timeliness and completeness revealed gaps for clinical form and transmission mode determination (p < 0.05), while increases in probable cases followed decreases in confirmed cases revealing gaps in laboratory diagnostics. Positive social change necessitates multidisciplinary collaboration between climatologists, clinicians, and epidemiologists to reach high-risk populations and promote educational awareness. The potential for social change includes predictive modeling optimizing funding while representing underserved populations.

<p><i>Francisella tularensis</i> is a Gram-negative intracellular pathogen causing the zoonotic disease tularemia. <i>F. tularensis</i> can be found almost all over the world and has been recovered from several animal species, even though the natural reservoir of the bacterium and parts of its life cycle are still unknown. Humans usually get infected after handling infected animals or from bites of blood-feeding arthropod vectors. There are four subspecies of <i>F. tularensis</i>: the highly virulent <i>tularensis</i> (Type A) that causes a very aggressive form of the disease, with mortality as high as 60% if untreated, the moderately virulent <i>holarctica</i> (Type B) and <i>mediasiatica</i>, and the essentially avirulent subspecies <i>F. novicida</i>. So far, our knowledge of the molecular mechanisms that would explain these differences in virulence among the subspecies is poor. However, recent developments of genetic tools and access to genomic sequences have laid the ground for progress in this research field. Analysis of genome sequences have identified several regions that differ between <i>F. tularensis</i> subspecies. One of these regions, RD19, encodes proteins postulated to be involved in assembly of type IV pili (Tfp), organelles that have been implicated in processes like twitching motility, biofilm formation and cell-to-cell communication in pathogenic bacteria. While there have been reports of pili-like structures on the surface of <i>F. tularensis</i>, these have not been linked to the Tfp encoding gene clusters until now. Herein, I present evidence that the <i>Francisella</i> pilin, PilA, can complement pilin-like characteristics and promote assembly of fibers in a heterologous system in <i>Neisseria gonorrhoeae. pilA</i> was demonstrated to be required for full virulence of both type A and type B strains in mice when infected via peripheral routes. A second region, RD18, encoding a protein unique to <i>F. tularensis</i> and without any known function, was verified to be essential for virulence in a type A strain. Interestingly, the non-licensed live vaccine strain, LVS (Type B), lacks both RD18 and RD19 (<i>pilA</i>) due to deletion events mediated by flanking direct repeats. The loss of RD18 and RD19 is responsible for the attenuation of LVS, since re-introducing them <i>in cis</i> could restore the virulence to a level similar to a virulent type B strain. Significantly, these deletion events are irreversible, preventing LVS to revert to a more virulent form. Therefore, this important finding could facilitate the licensing of LVS as a vaccine against tularemia.</p>

Francisella tularensis is a Gram-negative intracellular pathogen causing the zoonotic disease tularemia. F. tularensis can be found almost all over the world and has been recovered from several animal species, even though the natural reservoir of the bacterium and parts of its life cycle are still unknown. Humans usually get infected after handling infected animals or from bites of blood-feeding arthropod vectors. There are four subspecies of F. tularensis: the highly virulent tularensis (Type A) that causes a very aggressive form of the disease, with mortality as high as 60% if untreated, the moderately virulent holarctica (Type B) and mediasiatica, and the essentially avirulent subspecies F. novicida. So far, our knowledge of the molecular mechanisms that would explain these differences in virulence among the subspecies is poor. However, recent developments of genetic tools and access to genomic sequences have laid the ground for progress in this research field. Analysis of genome sequences have identified several regions that differ between F. tularensis subspecies. One of these regions, RD19, encodes proteins postulated to be involved in assembly of type IV pili (Tfp), organelles that have been implicated in processes like twitching motility, biofilm formation and cell-to-cell communication in pathogenic bacteria. While there have been reports of pili-like structures on the surface of F. tularensis, these have not been linked to the Tfp encoding gene clusters until now. Herein, I present evidence that the Francisella pilin, PilA, can complement pilin-like characteristics and promote assembly of fibers in a heterologous system in Neisseria gonorrhoeae. pilA was demonstrated to be required for full virulence of both type A and type B strains in mice when infected via peripheral routes. A second region, RD18, encoding a protein unique to F. tularensis and without any known function, was verified to be essential for virulence in a type A strain. Interestingly, the non-licensed live vaccine strain, LVS (Type B), lacks both RD18 and RD19 (pilA) due to deletion events mediated by flanking direct repeats. The loss of RD18 and RD19 is responsible for the attenuation of LVS, since re-introducing them in cis could restore the virulence to a level similar to a virulent type B strain. Significantly, these deletion events are irreversible, preventing LVS to revert to a more virulent form. Therefore, this important finding could facilitate the licensing of LVS as a vaccine against tularemia.

Background Indigenous peoples with traditional lifestyles worldwide are considered particularly vulnerable to climate change effects. Large climate change impacts on the spread of infectious vector-borne diseases are expected as a health outcome. The most rapid climate changes are occurring in the Arctic regions, and as a part of this region northernmost Sweden might experience early effects. In this thesis, climate change effects on the lives of Sami reindeer herders are described and 30 years of weather changes are quantified. Epidemiology of the climate sensitive human infection tularemia is assessed, baseline serologic prevalence of tularemia is investigated and the disease burden is quantified across inhabitants in the region. Methods Perceptions and experiences of climate change effects among the indigenous Sami reindeer herders of northern Sweden were investigated through qualitative analyses of fourteen interviews. The results were then combined with instrumental weather data from ten meteorological stations in a mixed-methods design to further illustrate climate change effects in this region. In two following studies, tularemia ecology and epidemiology were investigated. A total of 4,792 reported cases of tularemia between 1984 and 2012 were analysed and correlated to ecological regions and presence of inland water using geographical mapping. The status of tularemia in the Swedish Arctic region was further investigated through risk factor analyses of a 2012 regional outbreak and a cross-sectional serological survey to estimate the burden of disease including unreported cases. Results The reindeer herders described how the winters of northern Sweden have changed since the 1970s – warmer winters with shorter snow season and cold periods, and earlier spring. The adverse effects on the reindeer herders through the obstruction of their work, the stress induced and the threat to their lifestyle was demonstrated, forcing the reindeer herders towards the limit of resilience. Weather data supported the observations of winter changes; some stations displayed a more than two full months shorter snow cover season and winter temperatures increased significantly, most pronounced in the lowest temperatures. During the same time period a near tenfold increase in national incidence of tularemia was observed in Sweden (from 0.26 to 2.47/100,000 p&lt;0.001) with a clear overrepresentation of cases in the north versus the south (4.52 vs. 0.56/100,000 p&lt;0.001). The incidence was positively correlated with the presence of inland water (p&lt;0.001) and higher than expected in the alpine and boreal ecologic regions (p&lt;0.001). In the outbreak investigation a dose-response relationship to water was identified; distance from residence to water – less than 100 m, mOR 2.86 (95% CI 1.79–4.57) and 100 to 500 m, mOR 1.63 (95% CI 1.08–2.46). The prevalence of tularemia antibodies in the two northernmost counties was 2.9% corresponding to a 16 times higher number of cases than reported indicating that the reported numbers represent only a minute fraction of the true tularemia. Conclusions The extensive winter changes pose a threat to reindeer herding in this region. Tularemia is increasing in Sweden, it has a strong correlation to water and northern ecoregions, and unreported tularemia cases are quite common.

Tick-borne diseases are caused by a group of pathogenic microorganisms which are transmitted between animal and human population by vector which is most frequently tick. Evaluation of danger and perils of these diseases is important due to prevention and monitoring of current situation development. The most common tick-borne diseases in the Czech Republic are borreliosis, tick-borne encephalitis and ehrlichiosis. Rarely can we encounter bartonellosis, babesiosis, rickettsiosis and tularemia. All these diseases can have serious consequences and in critical cases they can result in death of the infected person. One of the basic and the most important of preventive measures which lower the risk of the infection by any tick-borne disease is prevention of tick encounter and eventually vaccination. Due to continual global warming we can expect that in near future ticks will spread even to locations which have been so far not suitable for their development. We can expect that we will even more frequently encounter tick-borne diseases - including diseases which were formerly not found in our territory or were very rare. As a part of health education it will be needed more to get known basic information of tick-borne diseases and their prevention. Different educational centres are an ideal place for...

La tularémie est une zoonose liée à la bactérie Francisella tularensis, hautement pathogène pour l’homme. La sous espèce la plus virulente, F. tularensis subsp. tularensis, est retrouvée uniquement en Amérique du Nord, alors que la sous-espèce F. tularensis subsp. holarctica est présente dans tout l’hémisphère Nord. En France toutes les souches appartiennent au biovar I de la sous-espèce holarctica et plus précisément au groupe phylogénétique B.FTNF002-00. Bien que rarement grave en France, la tularémie pose le problème de taux d’échecs thérapeutiques élevés, jusqu’à 25% en cas de traitement par ciprofloxacine ou gentamicine, et 35% pour la doxycycline. Les causes de ces échecs ne sont pas bien élucidées à l’heure actuelle. L’analyse de la littérature ainsi que la détermination de la sensibilité de 59 souches françaises de F. tularensis subsp. holarctica à 18 antibiotiques, confirment qu’aucune souche isolée à ce jour ne présente de résistance acquise à ces trois familles d’antibiotiques, qui représentent le traitement de première ligne de la tularémie. Les fluoroquinolones (en particulier la ciprofloxacine et la lévofloxacine) présentent concentrations minimales inhibitrices les plus basses, devant la gentamicine et la doxycycline. Les données disponibles in vitro et en modèle animal étant corrélées aux données humaines en termes d’efficacité et de taux d’échecs thérapeutiques, il semble néanmoins préférable de positionner la ciprofloxacine en première ligne pour le traitement des formes modérées de tularémie et de limiter l’utilisation de la doxycycline aux cas de contre-indication aux fluoroquinolones. L’azithromycine et la télithromycine ont été identifiées comme des alternatives thérapeutiques envisageables en cas d’infection par une souche de biovar I de F. tularensis subsp. holarctica lorsqu’existe une contre-indication aux traitements de première ligne. Des études en modèles animaux restent néanmoins nécessaires pour conforter ces dernières observations. La sélection in vitro de souches résistantes aux fluoroquinolones est possible, ce qui suggère la possibilité d’émergence de mutants résistants in vivo pour expliquer les taux d’échec thérapeutiques. Les principales mutations de résistance aux fluoroquinolones chez F. tularensis sont observées au niveau des gènes gyrA et gyrB codant pour les topoisomérases de type II. L’impact fonctionnel de mutations de résistances aux fluoroquinolones a été caractérisé in vitro chez F. novicida, pris comme modèle de bactérie avirulente proche de F. tularensis. L’activité de superenroulement et de clivage de l’ADN en présence de fluoroquinolones a été déterminée suite à la reconstruction in vitro de complexes GyrA/GyrB fonctionnels. La résistance aux fluoroquinolones était la plus forte en cas de mutation D87G/D87Y pour la sous-unité GyrA ou +P466 pour la sous-unité GyrB. La mutation P43H située en dehors du QRDR de GyrA est à l’origine d’un plus faible niveau de résistance. La mutation D487R-∆K488 en dehors du QRDR de GyrB ne confère pas de résistance intrinsèque mais potentialise l’effet d’une mutation D87G concomitante. En revanche, l’identification de mutations de résistance in vivo au sein des QRDR des gènes gyrA et gyrB chez des patients en situation d’échec thérapeutique traités par une fluoroquinolone est demeurée négative. Enfin, notre recherche a permis d’identifier de nouveaux composés de synthèse de structure bis-indolique possédant des activités antibactériennes. Ces composés sont bactériostatiques vis-à-vis de F. tularensis mais bactéricides vis-à-vis des staphylocoques y compris vis-à-vis de souches multi-résistantes de Staphylococcus aureus avec des CMI90 évaluées à 2mg/L chez F. tularensis et S. aureus pour le composé le plus actif. La faible solubilité de ces composés en milieu aqueux, leur forte liaison aux protéines plasmatiques ainsi que la recherche de leur mécanisme d’action original appellent néanmoins de nombreux développements futurs. / Tularemia is a zoonosis caused by the highly pathogenic bacterium Francisella tularensis. The most virulent subspecies, F. tularensis subsp. tularensis, is found only in North America while the subspecies F. tularensis subsp. holarctica is present in the whole Northern hemisphere. In France, all strains belong to the biovar I of the subspecies holarctica and more specifically to the phylogenetic subclade B.FTNF002-00. Although tularemia is usually not a severe disease in France, many patients suffer from therapeutic failures despite receiving an appropriate treatment. These treatments failures are observed in up to 25% of patients treated with ciprofloxacin or gentamicin, and up to 35% if patients treated with doxycycline. The causes of those therapeutic failures remain poorly elucidated. Analysis of the literature and determination of the susceptibility of 59 French F. tularensis subsp. holarctica strains to 18 antibiotics confirmed that to date, no strain with acquired resistance to any of the first-line antibiotics used for treatment of tularemia have been isolated. The fluoroquinolones (in particular ciprofloxacin and levofloxacin) exhibit the lowest minimal inhibitory concentrations, compared to gentamicin and doxycycline. Data obtained in vitro and in animal models are concordant with human data concerning the efficacy of antibiotics and therapeutic failure rates. Thus, we advocate the use of ciprofloxacin as first-line treatment for mild form of tularemia, and the use of doxycyclin only as a second-line treatment in patients with contraindications to fluoroquinolones. Azithromycin and telithromycin may also be considered as potential therapeutic alternatives for tularemia cases caused by biovar I strains of the susbspecies holarctica, but only for patients with contraindications to first-line antibiotics. Further data in animal models are however required to consolidate our in vitro data. The in vitro selection of fluoroquinolone-resistant strains of F. tularensis has been reported. This suggests that the in vivo selection of such resistant mutants may occur. In vitro, the main fluoroquinolone resistance mutations occur in the gyrA and gyrB genes that encode type II topoisomerases of F. tularensis. We have characterized the functional impact of such mutations in avirulent F. novicida strains, taken as a surrogate of F. tularensis. Supercoiling and DNA cleavage activity of GyrA/GyrB complexes reconstituted in vitro have been determined in the presence of fluoroquinolones. Fluoroquinolone resistance level was the highest in strains with a D87G/D87Y mutation in the GyrA subunit or +P466 mutation in the GyrB subunit. The mutation P43H located outside the GyrA Quinolone-Resistance-Determining-Region (QRDR) confered significant but lower fluoroquinolone resistance. The mutation D487R-∆K488 also outside GyrB QRDR did not cause fluoroquinolone resistance by itself, but increased the resistance level in case of concomitant D87G mutation. No mutation could be identified in vivo in the QRDR of gyrA and gyrB genes amplified from clinical samples collected in patients treated with a fluoroquinolone, although some of them experienced therapeutic failure. Finally, while searching for new antibiotic compounds, we identified new synthetic bis-indolic derivatives with antibacterial activity. Lead compounds were only bacteriostatic against F. tularensis but bactericidal against staphylococci including against multi-drug-resistant Staphylococcus aureus. MIC90 were measured at 2mg/L for F. tularensis and S. aureus strains for the most active compound. However, many developments are still required to improve their solubility in water, decrease their plasma proteins binding and elucidate their original mechanism of action.

Francisella tularensis is a highly infectious. Gram-negative coccobacillus that is the etiological agent of the acute. febrile. zoonotic disease tularemia. A ca. 35 kb Francisella pathogenicity island (FM) was previously discovered. Two genes. pdpA and pdpD were shown to be required for virulence. The FP1 gene pdpC encodes a protein that has no significant similarities to any motifs, domains, or homologues of known bacterial proteins. This gene of unknown function may encode a novel virulence factor involved in Francisella infection. The role of PdpC in F. novicida intracellular growth was investigated. Results from this study demonstrated that the erythromycin allelic replacement mutant of pdpC was more attenuated in intracellular growth in the murine macrophage-like J774A.1 cells than in bone marrow-derived macrophages from BALB/c mice and that complementation in trans partially complements this mutation. To further investigate the role of pdpC in virulence. partial deletion mutagenesis in the C-terminus of PdpC was performed which resulted in four mutants that showed slight attenuation in J774A.1 intramacrophage growth but behaved like wildtype F, novicida in bone marrow-derived macrophages. Chicken embryos were infected to evaluate the virulence of these pdpC mutants. The virulence of the Em allelic replacement mutant was significantly more attenuated than wildtype F. novicida and complementation partially restored virulence. Partial deletion mutants of pdpC exhibited greater virulence than the EmR mutant in chicken embryos and were able to cause 100% mortality at day 6. Furthermore, eukaryotic expression of triple FLAG-tagged PdpC in chicken embryo fibroblasts resulted in cells that exhibited different morphologies than uninfected fibroblasts which suggests that PdpC may play a role in cytoskeletal rearrangements by altering host cell signaling pathways.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb 17, 2009). Includes bibliographical references.

Francisella tularensis is a highly infectious. Gram-negative coccobacillus that is the etiological agent of the acute. febrile. zoonotic disease tularemia. A ca. 35 kb Francisella pathogenicity island (FM) was previously discovered. Two genes. pdpA and pdpD were shown to be required for virulence. The FP1 gene pdpC encodes a protein that has no significant similarities to any motifs, domains, or homologues of known bacterial proteins. This gene of unknown function may encode a novel virulence factor involved in Francisella infection. The role of PdpC in F. novicida intracellular growth was investigated. Results from this study demonstrated that the erythromycin allelic replacement mutant of pdpC was more attenuated in intracellular growth in the murine macrophage-like J774A.1 cells than in bone marrow-derived macrophages from BALB/c mice and that complementation in trans partially complements this mutation. To further investigate the role of pdpC in virulence. partial deletion mutagenesis in the C-terminus of PdpC was performed which resulted in four mutants that showed slight attenuation in J774A.1 intramacrophage growth but behaved like wildtype F, novicida in bone marrow-derived macrophages. Chicken embryos were infected to evaluate the virulence of these pdpC mutants. The virulence of the Em allelic replacement mutant was significantly more attenuated than wildtype F. novicida and complementation partially restored virulence. Partial deletion mutants of pdpC exhibited greater virulence than the EmR mutant in chicken embryos and were able to cause 100% mortality at day 6. Furthermore, eukaryotic expression of triple FLAG-tagged PdpC in chicken embryo fibroblasts resulted in cells that exhibited different morphologies than uninfected fibroblasts which suggests that PdpC may play a role in cytoskeletal rearrangements by altering host cell signaling pathways.