Factors affecting a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Erwinia carotovora subsp. atroseptica were investigated. Optimum reaction conditions for detecting known cell numbers of Eca were found to be 2.μg/ml of coating γ-globulin and 1:4-00 enzyme-γ-globulin conjugate dilution. These conditions were determined using antiserum produced against glutaraldehyde-fixed, whole bacterial cells of strain E82 of Eca (serogroup I), and polystyrene microtitration plates (Dynatech substrate plates). In spite of these optimized conditions, variability was observed between sets of data obtained under identical experimental conditions. In order to minimize or eliminate this variability, different parameters were investigated. The washing procedure was standardized by the use of a controlled pressure-washing system employing distilled water, and two 15-sec washes at 34.4-8 kPa (5 psi), with 180° rotation of the plate between each wash. Tween-20 was eliminated from the washing solution, since it interferred with the sensitivity of the assay. This effect could not be related to the age of the Tween-20 employed. Well to well variability was observed with the polystyrene microtitration plates employed but it was not exclusive to the outside rows. The pattern of distribution of the "odd" wells within a plate changed, and the number of "odd" wells decreased with time. The maximum variation from the mean also decreased with time. Addition to different wells of an extra 5% of coating y-globulin, sample, and enzyme-y-globulin conjugate individually or in different combinations, failed to reproduce the variability observed thereby eliminating pipetting errors as a source of variability. The A[sub=+.05] values were influenced by the buffer solutions employed for sample and conjugate dilution. Any given buffer had a greater effect when used for conjugate dilution. The complete buffer of phosphate buffered saline (PBS)+ 0.05% Tween-20 +2.0% polyvinylpyrrolidone+0.2% egg albumin commonly used in virus work, was found to be suitable for the Eca system although its efficiency in the presence of plant material containing bacteria remains to be evaluated. This ELISA for Eca employing optimized coating and conjugate, a standardized washing procedure and a complete buffer for samples and conjugate dilution, routinely detected 10⁵ to 10⁶ cells/ml of only serogroup I of Eca when pure cultures of both homologous and heterologous strains were tested. At concentrations >10⁷ cells/ml, strains from serogroups XVIII, XX, and XXII of subsp. atroseptica and a few strains from serogroups II, III, IV, and V of subsp. carotovora also reacted. Even at high bacterial concentration (10° cells/ml) no cross reactions were observed with Pseudomonas marginalis and Corynebacterium sepedonicum. Heat treatment of cell suspensions of serogroup I at 60 C for 3-6 min enhanced A[sub=+.05] values but the level of sensitivity was not reduced below 105 cells/ml. Cross reactions with strains of subsp. caro- tovora serogroups III and V, observed at 10 cells/ml, were reduced but not eliminated by this heat treatment. Both heat-labile and heat-stable water-soluble antigens were detected by this ELISA for Eca. The media upon which cells were grown also affected the A[sub=+.05] values but this effect was not proportional to the amount of growth observed. Based on these results it was concluded that until well to well variability is eliminated, and sensitivity increased, there will be little incentive to use the double sandwich ELISA technique with plant sap where a reduction in sensitivity is likely. At this point ELISA seems to have little potential in routine surveys for detecting latent blackleg infections in certified seed potatoes. / Land and Food Systems, Faculty of / Graduate

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Erwinia strains from several geographic regions and several hosts were evaluated for virulence, sensitivity to siderophores produced by Pseudomonas fluorescens (Pf) and bacteriocins produced by Erwinia, and for the presence of plasmids. Selection of virulent strains of Erwinia for use in plant breeding programs for resistance to disease might be based upon quantitative characters which are correlated with disease severity rather than the biochemical reactions used to distinguish Erwinia carotovora subspecies. Quantitative assays for motility, polygalacturonate degradation, potato tuber infection, and tuber decay revealed that motility was correlated with infection (r = 0.83, p = 0.01) and polygalacturonate degradation with decay (r = 0.84, p = 0.01) of potato tubers. Siderophores produced by Pf and bacteriocins produced by Erwinia yielded variable results in bioassays against the Erwinia strains studied. Six of the 12 strains of Erwinia tested were resistant to Pf siderophores. The growth inhibition of sensitive strains was bacteriostatic and reversible by addition of iron (Fe II or Fe III) to the culture medium. Additionally, only one strain of Erwinia was sensitive to the bacteriocins produced by the other 12 strains. The resistance of Erwinia strains to Pf siderophores and Erwinia bacteriocins severely limits the potential for widespread application of these agents in biological control of Erwinia. Bacteriocin-like structures were detected in culture extracts of all 12 Erwinia strains studied. The presence of bacteriocins is indirect evidence that these strains harbor plasmids. Bacteriocin-coding plasmids may be the source of genetic and phenotypic variability demonstrated by the erwinias. The status and value of subspecific classification of Erwinia carotovora may therefore require re-evaluation.

No description available.

Master of Dental Surgery / History has recorded the antiquity of serious infections in the region of the head and neck. Today, our community still experiences major life-threatening infections in these anatomical locations, which pose significant management difficulties to the oral and maxillofacial surgeon. The aim of this thesis is to review the aetiology, diagnosis and treatment of some bacterial infections involving structures of the head and neck. Such infections may spread, causing serious complications with severe morbidity and occasionally death. This theses deals only with infections of bacterial origin and does not attempt to cover viral, or fungal agents or the chronic specific diseases of tuberculosis and syphilis, and makes no attempt to address the old question of focal infection. The literature review relates especially to Ludwig’s Angina which was first described so dramatically in 1836. To this day it remains as a clinically potentially lethal disease despite the progress of modern medicine. Numerous descriptions in the literature warn of the rapid appearance of symptoms and the danger of respiratory obstruction when management of the airway is not satisfactorily undertaken. Both odontogenic and non-odontogenic causes of orofacial and neck infections are reviewed. Odontogenic problems are given special emphasis as they are now of major concern. The significance of the potential fascial spaces in the face and neck which allow the spread of dental infections is also highlighter. A thorough knowledge of these anatomical relationships is still of the utmost importance to the surgeon if he is to be successful in treatment. The principle of surgical drainage of pus is as important in 1990 as it was 150 years ago. The biological basis for the onset and progress of such fulminating infections in the head and neck region is still poorly understood. One constant need is that the bacteria, both aerobic and anaerobic, be correctly identified. Microbiological techniques are constantly improving and provide an important adjuvant investigation, which then allows the surgeon to provide the most appropriate antimicrobial therapy. Principal to the many aspects of treatment is the ability to maintain the airway of the patient and to provide the depth of anaesthesia necessary to undertake the required surgery. Major bacterial orofacial infections may have severe local and far-reaching systemic effects. Such complications are discussed in all their ramifications. It should be realised that the presentation of these patients at a late stage, when complications have already supervened, may make diagnosis difficult. There is always a necessity to ensure that the underlying cause of the disease is accurately defined and that complication are not allowed to progress further. Finally, a retrospective study of the management of 90 patients with major bacterial orofacial infections who have been treated at Westmead Hospital is presented. The outcome of this study of some major bacterial orofacial infections of the head and neck is the need to stress the importance of urgent surgical management and maintenance of the airway, together with the microbiological determination of the causative organisms and their sensitivities, so that other than empirical antibiotics can be instituted early. This must be combined with an upgrading of the patients’ medical and dental status. It was demonstrated that, in the majority of these patients, ignorance and fear combined with a lack of routine dental care resulted in major infections arising from relatively simple odontogenic causes such as dental caries, periodontal disease and pericoronal infection related to impacted teeth. Without doubt, the immediate care of these patients demanded intensive management. However, it is important to recognise that dental education forms an integral part not only of the recovery programme for the afflicted patient, but also as a community health preventive measure of profound significance.

In broiler operations, various health problems develop during the final two weeks of the growing period, resulting in increased mortality and condemnation losses. At this stage, sickly birds were found to be systemically infected by various bacteria regardless of varied clinical signs. The main objective of the present study was to determine the prevalence and nature of systemic bacterial infections in unthrifty commercial broiler chickens and to establish a reproducible infection model in the laboratory. Thirty-one unthrifty 6-week-old broilers were obtained from three farms, and bacterial isolations were conducted on blood, liver, and hock joint. Bacteria were isolated from 87, 90, and 71% of the blood, liver and hock joint samples, respectively. Mean bacterial counts (log������ CFU/ml or g) of the blood and liver were 2.15 and 2.93, respectively. Among 132 bacterial isolates, major species were; Staphylococcus (60%), Corynebacterium (18%), Escherichia coli (5%), and Stomatococcus (4%). Among 79 Staphylococcus isolates, 77 were coagulase-negative. Major species of staphylococci were; S. lentus (19%), S. simulans (18%), S. cohnii (13%), S. gallinarum (10%) and S. captis (7%). In addition, 6 species of gram-positive and 5 species of gram-negative organisms were isolated. Apparently systemic infections were not caused by predominant pathogenic bacterial species, and adequately described as mixed infections. However, there were some significant relationships between isolated bacterial species and sampling sites, suggesting that certain organisms were abundant in the environment of a particular poultry house. These results indicate that systemic infections in market age broilers are caused by mixed bacterial species and suggest that they are caused by suppressed host antibacterial systems rather than pathogenic factors of microorganisms. Antibiotic susceptibility results showed 100% susceptibility of staphylococcal isolates (n=69) against vancomycin and enrofloxacin. Of these coagulase negative staphylococci showed 19% and 73% resistance against methicillin and penicillin G, respectively. There was also heterogeneity in antibiogram profiles within species of coagulase-negative staphylococci. Pathogenicity of representative field isolates from the above described study was tested in 5-day-old embryonated eggs and in 3- week-old broiler chicks. Consistent lethality was demonstrated with S. aureus in embryos. Staphylococcus intermedius or S. lentus demonstrated some pathogenicity, while S. gallinarum or Corynebacterium were non-pathogenic in embryos. In 3-week-old broilers, however, only S. aureus caused septicemia and death; other bacterial species mentioned above caused neither clinical signs of acute or chronic staphylococcosis nor mortality. / Graduation date: 1998

The crystal structure of ATP-dependent phosphoenolpyruvate carboxykinase (ATP-oxaloacetate carboxy-lyase, (transphosphorylating), E.C. 4.1.1.49; PCK) from Escherichia coli K12 has been determined using a combination of multiple isomorphous replacement and density modification, and refined to a R-index of 0.202 (R-free = 0.244) at 1.9 A resolution. PCK catalyses the decarboxylation and ATP-dependent phosphorylation of oxaloacetate to form phosphoenolpyruvate, the first committed step of gluconeogenesis in E. coli. Each PCK molecule consists of a 275 residue N-terminal and 265 residue C-terminal ar mononucleotide-binding domain, with the active site located within a cleft between the two domains. PCK is an open-faced, mixed $\alpha/\beta$ protein with a unique overall tertiary structure. The putative phosphate-binding region of the ATP-binding site adopts the P-loop motif common to many ATP- and GTP-binding proteins. However, the â-sheet topology of the mononucleotide-binding fold of PCK differs from all other families within the P-loop containing nucleoside triphosphate hydrolase superfamily, suggesting PCK represents the first member of a new family of such proteins. The mononucleotide-binding domain also differs in structure from the classical mononucelotide-binding fold (CMBF), common to adenylate kinase, RecA, p21$\sp{{Ha}-ras}$, and elongation factor-Tu. Several highly-conserved amino acid residues among the ATP-dependent PCK family, including R65, Y207, K212, K213, H232, K254, T255, D269, K288 and R333 appear to make up the active site of the enzyme. A cysteine residue, C233, is located near the active site, and in the E. coli enzyme this residue is buried and is probably not involved in substrate binding or catalysis. Previous chemical modification studies, on several ATP- and GTP-dependent PCKs, have been assessed in view of these structural results. A mechanism of catalysis based on these and additional results is proposed. The structure of E. coli PCK complexed with the calcium-analogue Tb$\sp{3+}$ has been refined to an R-index of 0.205 (R-free = 0.259) at 2.5 A. Two binding sites for Tb$\sp{3+}$ have been determined, one within the active site coordinating to the side chains of K213, H232, and D269, and the other within the C-terminal domain, coordinating to the side chains of E508 and E511. No large structural movements are observed in PCK as a result of Tb$\sp{3+}$ binding, even though Ca$\sp{2+}$ is a known activator.

Thesis (Ph. D.)--Open University. BLDSC no. DXN075774.

Master of Dental Surgery / History has recorded the antiquity of serious infections in the region of the head and neck. Today, our community still experiences major life-threatening infections in these anatomical locations, which pose significant management difficulties to the oral and maxillofacial surgeon. The aim of this thesis is to review the aetiology, diagnosis and treatment of some bacterial infections involving structures of the head and neck. Such infections may spread, causing serious complications with severe morbidity and occasionally death. This theses deals only with infections of bacterial origin and does not attempt to cover viral, or fungal agents or the chronic specific diseases of tuberculosis and syphilis, and makes no attempt to address the old question of focal infection. The literature review relates especially to Ludwig’s Angina which was first described so dramatically in 1836. To this day it remains as a clinically potentially lethal disease despite the progress of modern medicine. Numerous descriptions in the literature warn of the rapid appearance of symptoms and the danger of respiratory obstruction when management of the airway is not satisfactorily undertaken. Both odontogenic and non-odontogenic causes of orofacial and neck infections are reviewed. Odontogenic problems are given special emphasis as they are now of major concern. The significance of the potential fascial spaces in the face and neck which allow the spread of dental infections is also highlighter. A thorough knowledge of these anatomical relationships is still of the utmost importance to the surgeon if he is to be successful in treatment. The principle of surgical drainage of pus is as important in 1990 as it was 150 years ago. The biological basis for the onset and progress of such fulminating infections in the head and neck region is still poorly understood. One constant need is that the bacteria, both aerobic and anaerobic, be correctly identified. Microbiological techniques are constantly improving and provide an important adjuvant investigation, which then allows the surgeon to provide the most appropriate antimicrobial therapy. Principal to the many aspects of treatment is the ability to maintain the airway of the patient and to provide the depth of anaesthesia necessary to undertake the required surgery. Major bacterial orofacial infections may have severe local and far-reaching systemic effects. Such complications are discussed in all their ramifications. It should be realised that the presentation of these patients at a late stage, when complications have already supervened, may make diagnosis difficult. There is always a necessity to ensure that the underlying cause of the disease is accurately defined and that complication are not allowed to progress further. Finally, a retrospective study of the management of 90 patients with major bacterial orofacial infections who have been treated at Westmead Hospital is presented. The outcome of this study of some major bacterial orofacial infections of the head and neck is the need to stress the importance of urgent surgical management and maintenance of the airway, together with the microbiological determination of the causative organisms and their sensitivities, so that other than empirical antibiotics can be instituted early. This must be combined with an upgrading of the patients’ medical and dental status. It was demonstrated that, in the majority of these patients, ignorance and fear combined with a lack of routine dental care resulted in major infections arising from relatively simple odontogenic causes such as dental caries, periodontal disease and pericoronal infection related to impacted teeth. Without doubt, the immediate care of these patients demanded intensive management. However, it is important to recognise that dental education forms an integral part not only of the recovery programme for the afflicted patient, but also as a community health preventive measure of profound significance.

Thesis (Ph.D.) - James Cook University, 2004. / Typescript (photocopy)