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The mammalian intestine contains a dense and diverse community of microorganisms. The resident microbiota makes contributions to host to promote proper immune system development and limit pathogen colonization. In this study, the effects of microbiota disruption with or without TLRs stimulation on intestinal permeability and immunity were examined in C57BL/6 mice receiving antibiotic treatment for 6 days and in antibiotics-treated mice received dead E. coli or S. aureus at day 4. The results showed that antibiotic treatment significantly decreased the total number of bacteria including specific aerobic group Enterobacteriaceae and Enterococcus, and specific anaerobic group Lactococcus/Bifidobacterium in intestinal mucosa and lumen. Although only a slight increase in the intestinal permeability and no change in caspase-3 activity of intestinal mucosa were observed after antibiotic treatment, the bacterial translocation (BT) to mesenteric lymph nodes (MLN) increased significantly. Subsequent experiments showed that antibiotic treatment decreased the mucosal killing activity and the expression of non-defensin family including RegIII£], RegIII£^, CRP-ductin and RELM£] but not the defensin family, and increased the translocation of pathogen K. pneumoniae significantly, suggesting that the increase of BT to MLN after antibiotic treatment is likely due to a reduction in gut immunity rather than an increase of intestinal permeability. Moreover, stimulation of TLR4 reversed the effect of antibiotic treatment, suggesting that the functioning of TLR4 in intestinal epithelium is required to prevent pathogenic invasion and maintain intestinal homeostasis.

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2010. / Committee Chair: Patricia Sobecky; Committee Member: Eric Stabb; Committee Member: Jim Spain; Committee Member: Roger Wartell; Committee Member: Thomas DiChristina. Part of the SMARTech Electronic Thesis and Dissertation Collection.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Medicine / Master / Doctor of Medicine

The conditions by which the production, in liquid media, of hemolytic activity by growing cells of T. hyodysenteriae and T. innocens are described. / From a ribonucleic acid added culture broth of T. hyodysenteriae, two hemolysins are shown in the supernatant. For each of them, a purification scheme allowing the obtention of an homogeneous hemolytic preparation by gel filtration and electrophoresis, is proposed. One hemolysin is associated with ribonucleic acid (HN) while the later is associated with bovine serum albumin (HP). They share some properties: their synthesis is blocked by chloramphenicol, they do not have proteolytic or phospholipasic activities, they are insensitive to oxygen and their activity does not require bivalent cations or is sensitive to EDTA's action; they are lytic to all red blood cell's type tested. They differ by the size of their apparent molecular weight, their mode of action, the stability of their activity to temperature and different pH and, their pattern of sensitivity to proteolytic enzymes. / The isolation of a non-hemolytic mutant strain of T. hyodysenteriae and its use in a ligated ileal loop model in rabbit has shown that there is a relation between the loss of the hemolytic activity and the loss of the enterotoxic activity.

Under storage conditions of ambient temperature and relative humidity in South Africa, seed-associated mycoflora proliferates. Fusarium moniliforme is ubiquitous in newly-harvested maize, persisting for variable periods in storage, while Aspergillus flavus may represent the final group of species in the succession of aspergilli after grain storage under high temperature and/or high humidity. Many strains of these fungi produce toxigenic secondary metabolites (mycotoxins) under local storage conditions. Since pathogenic fungi may be present within the tissues of stored seeds, these contaminants will not be eradicated by external fungicide treatment, therefore a possible alternative is biological control. The aim of the present investigation was to ascertain whether certain strains and/or species of Trichoderma have potential as biocontrol agents against the seed-associated pathogenic fungi, Aspergillus flavus and Fusarium moniliforme. A study of the fungal growth in dual cultures revealed that from nine isolates of Trichoderma spp. (T harzianum and T viride), four had a noticeable inhibitory effect on the growth of the pathogenic fungi. Scanning electron microscopical investigation of fungal interaction demonstrated no obvious hyphal penetration by - Trichoderma spp. In addition, significant alteration of Fusarium hyphae, with pronounced collapse and loss of turgor, and production of aberrant conidial heads and microheads by A. flavus were observed. Evidence derived from some biochemical studies revealed that antibiosis (by production of extracellular enzymes, volatile compounds and possible antibiotics) is probably the mechanism involved in the antagonistic effect of the four aggressive Trichoderma spp. The in vitro studies demonstrated that the use of Trichoderma spp. as biocontrol agents against A. flavus and F. moniliforme appears promising. / Thesis (M.Sc.)-University of Natal, 1995.

The genus Bacillus is comprised of Gram-positive, rod-shaped, spore-forming bacteria which are well known for their ability to produce a diverse array of antimicrobial compounds. Ofparticular interest is the ability of certain strains to produce antifungal compounds. Such organisms have the potential for application in agriculture where they can be used as biocontrol agents against selected plant pathogenic fungi. A study was undertaken to further characterize selected Bacillus isolates that exhibit broad spectrum antifungal activity. Dual culture bioassays were used to screen seven selected Bacillus isolates for activity against four plant pathogenic fungi in vitro. All isolates were able to inhibit the pathogens to varying degrees. Two isolates, R29 and B81, were selected for further testing and characterization. Further bioassays were performed on five complex nutrient media which were adjusted to pH S.S and 7, and both incubated at 2SoC and 30°C" respectively. It was found that pH and media composition showed significant influences on the antifungal activities of the isolates tested, but that a SoC temperature difference in incubation temperature did not. Tryptone soy agar was found to give rise to the largest inhibition zones. Both isolates were tentatively identified using standard biochemical and morphological tests. Based on its phenotypic characteristics, R29 was identified as a strain of B. subtilis. B81 proved to be more difficult to assign to a specific group or species of Bacillus, though B. subtilis and B. licheniformis were considered to be the nearest candidates. Genomic DNA was extracted from both isolates and a portion of each of their 16s rDNA genes were amplified and sequenced for homology testing against the GeneBank database. Homology testing confirmed that both isolates were members of the genus Bacillus and most probably strains of B. subtilis. The DNA fragment used for sequencing proved to be too small to give conclusive identification of the isolates. Isolate R29 was selected for further characterization of its antifungal compound/so Growth curve studies using a defined synthetic medium showed that antifungal activity arose during the stationary phase and appeared to be closely linked to sporulation. The antifungal component of cell free culture supematant was extracted using various methods including thin layer chromatography, acid precipitation, hydrophobic interaction chromatography and methanol extractions. High performance liquid chromatography (HPLC) analysis of extracts from acid precipitation and hydrophobic interaction chromatography revealed two active peaks indicating that at least two antifungal compounds were produced. Methanol extracted samples produced the cleanest sample extract but only revealed one active peak from the HPLC fraction . Nuclear magnetic resonance analysis of purified samples indicated that the antifungal compound/s have aromatic complex and peptide structures. The extracted antifungal compounds were Protease K resistant and found to be thermostable at temperatures ranging 80-121oC, and, were active at pH ranges of 3-13. The antifungal compounds were found to exhibit similar properties to known antifungallipopeptides i.e. iturin A and fengycin A and B. Further characterization and identification of the active compounds is recommended usmg methods such as liquid chromatography mass spectrometer and matrix-assisted laser desorption ionisation time-of- flight. The results presented in this dissertation provide a basis from which antifungal compounds produced by strains ofBacillus can be further characterized. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Pseudomonas aeruginosa is an opportunistic human pathogen which, like many other Gram negative pathogens, employs quorum sensing - regulated virulence factors to establish an infection in its host. Quorum sensing in P. aeruginosa populations is controlled by low molecular weight (hapten) signalling molecules known as homoserine lactones (HSLs). Blocking bacterial communication using antibodies is an attractive strategy for infection control as QS takes a central role in P. aeruginosa infections, and antibodies can recognise their targets with exquisite specificity. There are two well-studied QS circuits in P. aeruginosa- the Las system and the Rhl system, controlled by two autoinducers compounds, 3-oxo-C12-HSL and C4-HSL respectively. Antibodies raised against HSL compounds can reduce the expression of virulence factors controlled by QS circuit and the immunomodulatory effects of 3- oxo-C12-HSL. In order to generate antibodies with high sensitivities against the autoinducer compounds of P. aeruginosa, a panel of HSL compounds was synthesised, conjugated to the carrier protein and used for sheep immunisation. High specificity anti-HSL antibodies were isolated from an immunised sheep antibody repertoire using phage display technology. These phage antibody hits were converted into single chain antibody (scAb) format, which possessed a HuCκ gene for detection and 6x histidine tag for purification. Soluble scAbs expressed in E. coli were purified and characterised using ELISA. Unique clones showing high sensitivity for free HSL compounds were reformatted into sheep-mouse chimeric IgGs, expressed transiently in COS 7 cells and characterised using biochemical assays. These cross-reactive monoclonal antibodies were shown to recognise HSL compounds in low nanomolar concentrations and have the potential to reduce virulence gene expression in P. aeruginosa.

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1979. / Bibliography: leaves 147-156. / Microfiche. / x, 156 leaves ill. (some col.) 29 cm