Bioterrorism

This book consists of nine chapters, written by international authorities, discussing various aspects of bioterrorism preparedness and response. Five of the chapters are agent-specific and highlight the pathogenesis, prevention and treatment, and the potential of specific organisms (Rickettsia and Yersinia pestis) or toxins (ricin, botulinum neurotoxins, and staphylococcal enterotoxins) to be used for nefarious purposes. Four chapters discuss different aspects of detecting and responding to a bioterrorism attack. These include methods for spatio-temporal disease surveillance, international laboratory response strategies, detection of botulinum neurotoxins in food and other matrices, and the use of physical methods (ie Raman spectroscopy) to detect spores

YERSINIA PESTIS-HOST CELL INTERACTIONS DURING DISTINCT INFLAMMATORY PHASES OF PRIMARY PNEUMONIC PLAGUE

Inhalation of Yersinia pestis causes primary pneumonic plague, one of the deadliest manifestations of plague. The pneumonic form of plague has played a critical role in the severity of both historical and modern plague outbreaks, yet the host-pathogen interactions that govern the lethality of Y. pestis pulmonary infections are incompletely understood. Primary pneumonic plague progresses in two distinct phases as defined by host immune responses and disease pathology. Inhaled Y. pestis colonizes the lungs and replicates to high numbers during the initial asymptomatic pre-inflammatory phase. After this time, disease progresses into the pro-inflammatory phase, and Y. pestis can be detected in the bloodstream and other organs. This second phase is characterized by the rapid onset of symptoms, high levels of pro-inflammatory cytokines in the lung, and a massive influx of neutrophils into the alveolar spaces. The goal of the research described in this dissertation was to characterize Y. pestis interactions with host cells and manipulations of the immune response during the different phases of disease. In Chapter 2, I used transposon sequencing to identify genes involved in Y. pestis adherence in the lung early after inoculation. I identified enrichment of genes involved in the regulation and assembly of macromolecules and determined that YPO3903, encoding a hypothetical protein, mediates adherence of Y. pestis wild-type following intranasal inoculation. In Chapter 3, I investigated bacterial burdens and IL-1 cytokine levels as factors initiating the pro-inflammatory switch. High Y. pestis lung burdens, but not bloodstream burdens or the changing ratio of pro- and anti-inflammatory IL-1 cytokines, trigger the onset of inflammation in the lung during primary pneumonic plague. In Chapter 4, I examined the effects of Y. pestis infection on neutrophil degranulation during the pro-inflammatory phase. I determined that Y. pestis inhibits neutrophil exocytosis of primary granules through direct type III secretion system translocation of effectors YopE and YopH. Taken together, these findings demonstrate the ability of Y. pestis to manipulate pulmonary immune defenses and cause severe disease in the lung.Doctor of Philosoph

Sex differences in immune protection in mice conferred by heterologous vaccines for pneumonic plague

BackgroundYersinia pestis is the etiological agent of plague, which can manifest as bubonic, septicemic, and/or pneumonic disease. Plague is a severe and rapidly progressing illness that can only be successfully treated with antibiotics initiated early after infection. There are no FDA-approved vaccines for plague, and some vaccine candidates may be less effective against pneumonic plague than bubonic plague. Y. pestis is not known to impact males and females differently in mechanisms of pathogenesis or severity of infection. However, one previous study reported sex-biased vaccine effectiveness after intranasal Y. pestis challenge. As part of developing a safe and effective vaccine, it is essential that potential sex differences are characterized. MethodsIn this study we evaluated novel vaccines in male and female BALB/c mice using a heterologous prime-boost approach and monitored survival, bacterial load in organs, and immunological correlates. Our vaccine strategy consisted of two subcutaneous immunizations, followed by challenge with aerosolized virulent nonencapsulated Y. pestis. Mice were immunized with a combination of live Y. pestis pgm- pPst-Δcaf1, live Y. pestis pgm- pPst-Δcaf1/ΔyopD, or recombinant F1-V (rF1-V) combined with adjuvants. ResultsThe most effective vaccine regimen was initial priming with rF1-V, followed by boost with either of the live attenuated strains. However, this and other strategies were more protective in female mice. Males had higher bacterial burden and differing patterns of cytokine expression and serum antibody titers. Male mice did not demonstrate synergy between vaccination and antibiotic treatment as repeatedly observed in female mice.ConclusionsThis study provides new knowledge about heterologous vaccine strategies, sex differences in plague-vaccine efficacy, and the immunological factors that differ between male and female mice

Temperature and pH Regulate PsaE and PsaF to Control Expression of psaA in Yersinia pestis

Yersinia pestis is a Gram-negative bacterial pathogen that causes the disease plague. Plague is a fulminant disease with a high mortality rate if left untreated, leading Y. pestis to be one of the deadliest pathogens in human history. Bubonic plague is the most common form of the disease in humans and many bacterial factors important for disease are poorly understood. The Y. pestis “pH 6 antigen” (PsaA) forms fimbriae and is important for bubonic plague pathogenesis. High temperature and low pH induce PsaA production and while the influence of these environmental cues on the production of PsaA is evident, the molecular mechanisms underlying this unusual regulation are not defined. Little attention has been focused on the regulation of psaA in Y. pestis and much of our current knowledge is based upon studies of psa in Y. pseudotuberculosis and the orthologous system, myf, in Y. enterocolitica. The focus of this dissertation was to characterize mechanisms Y. pestis utilizes to control psaA expression. Using defined growth conditions, we determined that psaA expression in Y. pestis is induced by high temperature and low pH and requires two genes, psaE and psaF, located directly upstream of psaA. We generated antibodies that recognize endogenous PsaE and PsaF and showed that temperature and pH control the levels of these proteins. High temperature is required for the translation of psaE and psaF and thus controls the production of PsaE and PsaF. Additionally, we identified a distinct pH threshold that defines PsaE and PsaF levels and identified residues in the periplasmic domains of each protein that enhance their stability. PsaF enhances PsaE levels. Histidine residues in the periplasmic domain of PsaF sense pH and thus, through its influence on PsaE stability, PsaF functions as a pH sensor that controls psaA expression. Together, our data indicate that Y. pestis utilizes temperature and pH to regulate the levels of PsaE and PsaF and precisely coordinate psaA expression. This work contributes to the understanding of molecular mechanisms that bacteria utilize to sense environmental variations and control gene expression.Doctor of Philosoph

CREATING A MULTIVALENT SUBUNIT VACCINE USING TYPE III SECRETION SYSTEM TIP PROTEINS AS ANTIGENS

Many gram-negative bacterial pathogens employ type III secretion systems (TTSS) to transport effector proteins into eukaryotic host cell membranes and cytoplasms to subvert normal cellular functions. TTSSs contain a basal body which spans the inner and outer bacterial membranes and a needle which extends the into extracellular space. With the increasing prevalence of drug resistant bacterial strains, vaccines represent one of the most promising strategies to combat these diseases. Proteins located at the extracellular needle tip of TTSSs appear to be excellent candidates for a sub-unit vaccine approach. These so called tip proteins are surface exposed and regulate the secretion of other effector proteins. This work presents pre-formulation, formulation and animal studies focused on creating a single multivalent sub-unit vaccine for five gram-negative pathogenic systems. In addition, a putative tip protein from Chlamydiae is compared to the other tip proteins using biophysical methods

Environmental Aspects of Zoonotic Diseases

Environmental Aspects of Zoonotic Diseases provides a definitive description, commentary and research needs of environmental aspects related to zoonotic diseases. There are many interrelated connections between the environment and zoonotic diseases such as: water, soil, air and agriculture. The book presents investigations of these connections, with specific reference to environmental processes such as: deforestation, floods, draughts, irrigation practices, soil transfer and their impact on bacterial, viral, fungal, and parasitological spread. Environmental aspects such as climate (tropical, sub-tropical, temperate, arid and semi-arid), developed and undeveloped countries, animal traffic animal border crossing, commercial animal trade, transportation, as well geography and weather on zoonosis, are also discussed and relevant scientific data is condensed and organized in order to give a better picture of interrelationship between the environment and current spread of zoonotic diseases

Properties of the cell surface of Aeromonas salmonicida

Merged with duplicate record 10026.1/562 on 08.03.2017 by CS (TIS)The properties of the cell surface of Aeromonas salmonicida were studied, with particular emphasis on the additional surface layer (A-layer), found on virulent strains. This was identified by electron microscopy, as having a tetragonal subunit morphology; and by electrophoresis of membrane components as a 51 kdal protein on A+ strains. A+ and A- strains, (the latter isolated by growth at elevated temperature), were compared biochemically and their interactions with various cell types investigated. Strains of A. salmonicida possessing the A-layer were shown to be more hydrophobic than those devoid of this protein. The influence of culture age, medium composition and subculture on hydrophobicity were investigated and hydrophobicity related to culture characteristics. No difference in enzyme susceptibility between A+ and A- A. salmonicida was found and both phenotypes showed similar tolerance to other environmental conditions. The interactions between A. salmonicida and cells in vitro were studied using adhesion and association assays involving radiolabelled bacteria, viable count determinations, haemagglutination and chemiluminescence. A+ A. salmonicida were found to adhere to a greater extent than A- bacteria to tissue culture cell lines and to isolated fish tissue by non-specific hydrophobic interactions. Adhesion was maximal to a fish epithelial cell line and the effects of various environmental conditions on adhesion were determined. A- bacteria more commonly exhibited haemagglutination which was inhibited by specific sugars. A+ bacteria associated more than A- organisms with mouse peritoneal macrophages and rainbow trout phagocytes when in salts solutions. The effects of opsonization on A. salmonicida were strain dependent. Incubation in a variety of sera resulted in a decrease in A+ surface hydrophobicity, often accompanied by abolition of characteristic autoagglutinability, whereas opsonized A- cells became more hydrophobic. These properties directly influenced the chemiluminescence response of trout macrophages.The Ministry of Agriculture, Fisheries and Food, Weymout

Étude des gènes d'Actinobacillus pleuropneumoniae exprimés en conditions d'infection

Actinobacillus pleuropneumoniae est l’agent étiologique de la pleuropneumonie porcine. La bactérie se transmet par voies aériennes et contacts directs. Plusieurs facteurs de virulence ont été identifiés, nommément les polysaccharides capsulaires, les lipopolysaccharide, les exotoxines ApxI à IV et de nombreux mécanismes d’acquisition du fer. Aucun vaccin efficace contre tous les sérotypes de la bactérie n’a encore été élaboré. Afin de mieux comprendre de quelle façon A. pleuropneumoniae régule la transcription de ses nombreux facteurs de virulence et de découvrir de nouvelles cibles potentielles pour l’élaboration de vaccins efficaces, le profil transcriptomique de la bactérie a été étudié dans des conditions simulant l’infection ainsi qu’à la suite d’une infection naturelle aiguë chez l’animal. Des biopuces de première et de seconde génération (AppChip1 et AppChip2) comportant respectivement 2025 cadres de lecture ouverts (ORF) de la version préliminaire du génome d’A. pleuropneumoniae sérotype 5b souche L20 et 2033 ORF de la version finale annotée du même génome ont été utilisées. Dans un premier temps, des expériences réalisées dans des conditions de concentration restreinte en fer ont permis d’identifier 210 gènes différentiellement exprimés, dont 92 étaient surexprimés. Plusieurs nouveaux mécanismes d’acquisition du fer ont pu être identifiés, incluant un système homologue au système YfeABCD de Yersinia pestis, impliqué dans l’acquisition du fer chélaté, ainsi que des gènes homologues aux composantes du système HmbR de Neisseria meningitidis impliqué dans l’acquisition du fer à partir de l’hémoglobine. Dans des conditions de culture permettant la formation de biofilms, les gènes tadC et tadD d’un opéron tad (« tight adherence locus ») putatif, les gènes pgaBC impliqués dans la synthèse d’un polysaccharide de la matrice du biofilm ainsi que deux gènes présentant de fortes homologies avec un gène codant pour l’adhésine auto-transporteur Hsf retrouvée chez Haemophilus influenzae ont montré une surexpression significative. Plusieurs de ces gènes ont également été retrouvés lors d’expériences réalisées avec des cellules épithéliales d’origine pulmonaire en culture, qui ont permis d’identifier 170 gènes différentiellement exprimés après la croissance planctonique au-dessus des cellules, et 131 autres suite à l’adhésion à ces cellules. Parmis les gènes surexprimés, les gènes tadB et rcpA de l’opéron tad putatif, les gènes pgaBC ainsi que le gène codant pour l’homologue d’Hsf ont été retrouvés. En présence de liquide de lavage broncho-alvéolaire (BALF), 156 gènes ont montré un profil d’expression modifié, et le gène apxIVA, identifié comme étant surexprimé, a pu être détecté pour la première fois dans des conditions de croissance in vitro. Finalement, des expériences visant à déterminer les gènes utilisés directement chez l’animal en phase aiguë de la pleuropneumonie porcine ont permis d’identifier 150 gènes qui étaient différentiellement exprimés. En plus d’identifier des gènes d’un possible opéron codant pour un fimbriae de type IV, 3 des 72 gènes surexprimés sont conservés chez tous les sérotypes d’A. pleuropneumoniae et codent pour des protéines ou lipoprotéines de surface. Nos expériences ont permis d’identifier plusieurs nouveaux facteurs de virulence potentiels chez A. pleuropneumoniae ainsi que plusieurs nouvelles cibles potentielles pour l’élaboration de vaccins efficaces contre tous les sérotypes.Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia. Transmission of the disease occurs through direct contact or aerosols. The bacteria possess many virulence factors, namely capsular polysaccharides, lipopolysaccharides, four Apx toxins and iron acquisition mechanisms. To this day, an efficient cross-serotype vaccine has yet to be developed. In order to investigate regulation mechanisms in A. pleuropneumoniae and to identify new potential targets for the synthesis of subunit vaccines, the transcriptomic profile of the bacteria under conditions that simulate the infection and following a natural acute infection in vivo were studied. The experiences relied on first and second generation microarrays (AppChip1 and AppChip2) designed using 2025 ORFs of the draft version of the A. pleuropneumoniae serotype 5b strain L20 genome and 2033 ORFs of the final and annotated version of the same genome respectively. First, experiments were conducted under iron-restricted conditions and 210 genes were deemed differentially expressed, 92 of which were up-regulated. Some new putative iron acquisition mechanisms were identified, including genes homologous to those of the Yersinia pestis YfeABCD chelated-iron acquisition system, as well as other genes homologous to components of the HmbR iron uptake from hemoglobin system of Neisseria meningitidis. When cultured in conditions promoting biofilm production, genes tadC and tadD from a putative tad (« tight adherence locus ») operon, genes pgaABC involved in the biosynthesis of a polysaccharide of the biofilm matrix as well as two ORFs encoding a putative autotransporter adhesins similar to the Haemophilus influenzae Hsf adhesin were all significantly overexpressed. Many of these genes were also overexpressed when lung epithelial cells were infected with A. pleuropneumoniae. While 170 genes were differentially expressed after planktonic growth in the culture medium above the cells, another 131 were identified following direct adhesion to the cells. Genes tadB and rcpA of the tad locus, as well as genes pgaBC and an ORF coding for the Hsf homolog where all found among overexpressed genes. When A. pleuropneumoniae was cultured in contact with broncho-alveolar lavage fluids (BALF), 156 genes were significantly differentially expressed and gene apxIVA, which was up-regulated, was detected for the first time during in in vitro growth conditions. Finally, experiments were conducted in vivo in animals naturally infected with A. pleuropneumoniae in the late stage of the acute phase in order to identify genes that are expressed during the infection of the natural host. While 150 genes were deemed differentially expressed, genes apxIVA as well as two genes from an operon coding for a putative type IV fimbriae were up-regulated. Out of those 72 genes that were overexpressed, 3 encode proteins or lipoproteins of the outer membrane which are conserved among all serotypes of the bacteria. Overall, we were able to identify several new potential virulence factors for A. pleuropneumoniae in the course of our experiments, as well as several new potential targets for the elaboration of an efficient cross-serotype vaccine