Vibrio cholerae, a natural inhabitant of aquatic environments and the causative agent of the diarrheal disease cholerae, requires iron for survival. Since one of the key factors in the survival of V. cholerae in the environment is the formation of biofilms, we determined the effect of iron on this aspect of the pathogens lifestyle. Since wild type V. cholerae forms a much more robust biofilm in the presence of exogenous iron we tested mutants in iron transport and regulation and found that a mutation in the gene encoding an iron-regulated small RNA, RyhB, was clearly attenuated in the biofilm assay. We determined through microarray analysis that the ryhB mutant has altered regulation of genes involved in many systems that may be involved in biofilm formation including amino acid biosynthesis, the TCA cycle, motility and chemotaxis, and the expression of outer membrane proteins. Due to the pleiotropic regulatory effects of RyhB, it is unlikely that any one individual gene or system regulated by RyhB is the cause of the biofilm defect, but rather the sum effect of the regulatory changes is decreased biofilm formation. Additionally, we discovered that the outer membrane protein, OmpT, is positively regulated by iron and Fur. Generally, when Fur has acted as a positive regulator in previous studies, it has been ultimately shown to do so by negatively regulating the negative regulator, RyhB. However, the positive regulation of ompT by Fur is independent of RyhB. While CRP, a positive regulator of ompT expression, did not affect iron-dependent regulation of ompT, over-expression of the negative regulator ToxR abolishes the iron and Fur dependent regulation. Sequence analysis has revealed a possible Fur box approximately 70 base pairs upstream of the transcriptional start site in a region that overlaps both a ToxR binding site and a CRP binding site in the ompT promoter. We propose the model that in iron-replete environments under ToxR repressing conditions, such as when amino acids are limiting, Fur can further increase the expression of ompT. / text

published_or_final_version / Criminology / Master / Master of Social Sciences

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 4 uppsatser.

Thesis (M. Soc. Sc.)--University of Hong Kong, 1997. / Includes bibliographical references (leaf 73-83).

Diss. (sammanfattning) Göteborg : Univ. , 2009. / Härtill 3 uppsatser.

Thesis (M.Soc.Sc.)--University of Hong Kong, 1997. / Includes bibliographical references (leaf 73-83) Also available in print.

To identify antigens unique to live Pasteurella multocida P1059, 10 week old specific pathogen-free (SPF) chickens were vaccinated three times with one of the following: viable cells from P. multocida P1059, 3865, 3866, or cells from formalin-killed strain PI059 or formalin-killed strain P1059 that were opsonized with antiserum directed against killed strain PI059 prior to immunization. Vaccinated birds were challenged with 1.5 x 10⁷ CFU of live strain P1059. Eight, 71, 86, and 50% of the birds that received live strains P1059, 3865, 3866 and killed strain P1059 (respectively), exhibited clinical signs of fowl cholera. Antisera directed against live strain PI059 recognized 23 proteins ranging from 14- to 92-kilodaltons (kDa); 20 of which were adsorbed by strain 3865. The molecular masses of the three remaining proteins were 25-, 30- and 43-kDa. A genomic library of strain P1059 was constructed using the plasmid vector pUC-19 and screened with antisera against live strain P1059; 12 out of 4,100 clones were recognized. The inserts of the plasmids from these clones ranged from 0.48- to 6.S-kilobases (kb) in length. Five of the 12 clones expressed proteins with molecular masses of 34-, 37-, 42-, 46- and 55-kDa. Escherichia coli CSR603(pOP43- 2G) and CSR603(pOP33-SF) expressed proteins recognized by antisera directed against live strain P1059. E. coli CSR603(pOP43-2G) expressed an epitope(s) which was recognized by antisera directed against strains 3865 and 3866. Conditions for transformation were optimized and attempts were made to create a shuttle vector in order to establish a cloning system for gene expression in P. multocida. The highest efficiency of transformation (1.25 x 10⁷ CFU/μg DNA) was obtained when 7.6 x 10¹⁰ cells of P. multocida R473 were electroporated at 12.5 kV cm⁻¹ for 10 ms with 5 ng of the plasmid, p VM109. Of the six strains tested, representing serogroups A, B, D and E, all were transformed successfully. Vectors including pBR322, pUC19, pJFF224-NX and pSP329 were unable to transform P. multocida. To create a shuttle vector for gene expression in P. multocida, a Pasteurella plasmid (pLAR-1) was cloned in both orientations into the BamH I site of pBR322. These plasmids, pLRBR-21 and pLRBR-67, had a transformation efficiency of 4.5 to 8 x 10⁴ CFU/μg of DNA in strain R473. Chromosomal DNA containing the Brucella abortus copper-zinc superoxide dismutase gene was cloned into the Cla I site of pLRBR-21. The 1.8-kb fragment encoding a 42-kDa Pasteurella protein was cloned into an additional unique site (Nru 1) of pLRBR-21 to determine if this plasmid was a viable shuttle vector for gene expression in P. multocida. / Ph. D.

D. Tech. (Department of Chemistry, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Cholera is a waterborne disease caused by toxigenic strains of Vibrio cholerae. The spread of cholera in developing countries has largely been imputed to the unavailability of proper water treatment and sanitary infrastructure as well as poor hygiene. In order to prevent the contraction and spread of cholera the use of solar disinfection (SODIS) to treat water in waterborne endemic communities has been recommended by the World Health Organization (WHO). SODIS is a water sterilizing method that relies on natural sunlight to improve the microbiological quality of water. During SODIS the culturability of the water contaminating microorganisms is inactivated by the ultraviolet component of solar radiation. The success of SODIS treatment of water in alleviating the risks associated with the contraction of waterborne diseases such as cholera has been attributed to the effectiveness, with which the water is treated, simple application as well as low cost of materials required. Currently SODIS research has been dominated by studies geared towards understanding how the microbial inactivation occurs, enhancement of the disinfection process and health impact assessments. However, little to no research has been directed towards exploring the role played by the immune system following the consumption of the solar irradiated water pathogens such as V. cholerae. SODIS of microorganisms in water results in immunologically important microbial states and components that could induce an immune reaction or response. In view of the role of dendritic cells in shaping an immune response, the effect that solar irradiated V. cholerae in water has on the immunochemistry of the dendritic cells in vitro was investigated. Prior to the stimulation of the dendritic cells with the solar irradiated cultures of V. cholerae, the first objective required an evaluation on the impact that solar irradiation has on the production and secretion of the cholera toxin by V. cholerae in water. The results from this evaluation showed that solar ultraviolet radiation was incapable of inducing the secretion of the cholera toxin. Furthermore, there was extensive DNA degradation in the solar irradiated cultures of V. cholerae. The second objective was to investigate the ability for solar irradiated cultures of V. cholerae in water to induce the phenotypic maturation of immature dendritic cells in vitro. In order to achieve this objective, solar and non-solar irradiated, chemically/ heat inactivated and phosphate buffered saline (PBS) prepared cultures of V. cholerae as well as lipopolysaccharide (LPS) and cholera toxin-β (CTB) subunit were each used to stimulate immature dendritic cells. After 48 hours of stimulation the dendritic cells were assessed for the expression of CD54, CD80, CD83, CD86, MHC-I and MHC-II on their cell membrane. The results showed an increase in the expression of all the maturation phenotypic markers with CD54, CD86 and MHC-I being the most prominent ones on the surface of the dendritic cells stimulated with solar irradiated cultures of V. cholerae. The third objective was to assess the profile of the cytokines and chemokines secreted by the dendritic cells following their stimulation with solar and non-solar irradiated, chemically/heat inactivated and PBS prepared cultures of V. cholerae as well as LPS and CTB subunit. After 48 hours of dendritic cell stimulation the tissue culture media from each treatment was quantitatively and qualitatively analysed for the presence of interleukin (IL)-1α, IL-1β, IL-6, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, IL-23, IL-27, macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) and tumor necrosis factor (TNF)-α. The analysis revealed that solar irradiated cultures of V. cholerae induced dendritic cells to secrete significant levels of pro-inflammatory cytokines in comparison to the unstimulated dendritic cells. Furthermore the profile of the cytokines and chemokines secreted by the dendritic cells in response to the solar irradiated cultures of V. cholerae in water was similar to that required to induce a T- helper (Th) Th2 immune response. The fourth objective was to assess the expression of the toll like receptor (tlr) genes by the dendritic cells following their stimulation with solar and non-solar irradiated, chemically/heat inactivated and PBS prepared cultures of V. cholerae as well as LPS and CTB subunit. After 48 hours of stimulation total RNA was extracted from the dendritic cells and subjected to real time quantitative polymerase chain reaction (RT qPCR) assay for tlr 1, 2, 3, 4, 5, 6, 9, 11, 12 and 13. The results showed no significant increase or decrease in the expression of most tlr genes in comparison to the unstimulated dendritic cells. This observation is synonyms with dendritic cell maturation. Taken together these findings show that solar irradiated cultures of V. cholerae were able to induce the maturation of immature dendritic cells in vitro. Furthermore dendritic cells stimulated with solar irradiated cultures of V. cholerae produced pro-inflammatory cytokines and chemokines. The results from this study suggests that the consumption of SODIS treated could provide immunological benefits.

Master of Science / Department of Horticulture, Forestry, and Recreation Resources / David A. Haukos / There is a wide variety of diseases that affect wild migratory birds. Occurrence, causes, and impacts of disease outbreaks in wild bird populations are rarely studied beyond documentation of large epizootic events. The relationships between the wildlife-livestock-human interface is rapidly blurring together. Global interests in avian diseases increased around 1990 as a result of the prevalence of zoonosis and potential threat to domestic livestock. A central disease reporting protocol does not exist in many states, which has led to a lack of available historical knowledge of disease occurrence that could be used to predict and manage future outbreaks. Due to changes of abundance and distribution of the migrant populations of Ross’s goose (Chen rossii) and Snow goose (C. caerulescens), geese are increasing their stopover duration in Kansas potentially increasing risk of disease outbreaks. We compiled historic records of wild waterfowl disease events in Kansas from 1967-2014 and related the frequency of events with indices of light geese abundance from 1970-2014. We found 32 reports spanning 16 counties consisting of the diseases avian cholera, avian botulism, aspergillosis, renal coccidiosis, west Nile, aflatoxicosis, and mycotoxicosis. Using a retrospective survey, we found there was a significant relationship between population densities of light geese in Kansas during the Mid-Winter Waterfowl Inventory and occurrence of avian cholera. Efforts to increase the understanding of relationships between disease outbreaks and host species will improve management of future disease outbreaks. Understanding factors known to facilitate wild waterfowl disease events (e.g., environmental, species, and individual), may assist in disease identification and determine a disease management course of action. This course of action is predetermined in a disease management plan. Disease management plans should be developed at the state and station level; incorporating planning, response, disease control, and surveillance and monitoring schemes to build upon the centralized disease database and to promote future disease understanding.

M.Tech. (Chemistry) / Please read abstract in the full-text document