Thesis (M.S.) in Botany and Plant Pathology--University of Maine, 2002. / Includes vita. Includes bibliographical references (leaves 67-72 ).

Thesis (M.S.)--University of Wisconsin--Madison, 1965. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S. in geology)--Washington State University, December 2006. / Includes bibliographical references (p. 55-60).

Thesis (M.S.) University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (June 27, 2007) Includes bibliographical references.

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The antibiotic, 2,4-diacetylphloroglucinol (Phl), is produced by a range of naturally isolated fluorescent pseudomonads, found in disease suppressive soils. The natural isolate, P. fluorescens F113, protects pea plants from the pathogenic fungus, Pythium ultimum, by reducing the number of pathogenic lesions on the plant's roots. This beneficial effect was however, outweighed by the F113 causing an overall reduction in the emergence of the pea plants in the infected soil. The gene locus responsible for the Phl production was shown to be functionally conserved between the P. fluorescens F113 and another Phl producing organism, P. fluorescens Q2-87. Following identification of this functional sequence homology, the genes were isolated from F113, by optimised, long PCR. The 6.7-kb gene cluster was inserted into the chromosome of a non-pathogenic P. fluorescens, SBW25, which can effect biological control against the plant pathogen, Pythium ultimum through competitive exclusion of the fungus, by means of its strong colonising competence. The insertion was a targeted, homologous recombination designed to insert the Phl coding genes, from the F113, into a non-essential, lacZY coding region of the SBW25 chromosome. The transformed strains of SBW25 assumed two different morphological appearances. The morphological changes were noted at a ratio of 1:1 of normal morphology and altered morphology. Transformation of SBW25 with the Phl locus without this repressor element led to transformants with only normal morphology. All transformants were able to suppress P. ultimum through antibiotic production following the Phl transformation. However, the fitness of the transformants was reduced in flask culture, at 30°C, against the un-transformed SBW25. The organisms transformed with the entire Phl locus were seen to clump together in the culture media. The strain transformed with the Phl locus lacking the repressor element behaved normally. When inoculated on pea seedlings, the strain containing no repressor element behaved similarly to the F113, causing lower pea seed emergence. A transformant containing the entire Phl genetic locus had not lost its environmental competence on the pea roots, maintaining a high population, but was unable to maintain a high population in the surrounding soil.

Foodborne illnesses following the ingestion of contaminated food are a major public health problem worldwide. They include a broad group of illnesses ranging from mild to chronic or life-threatening; caused by either toxins released from the disease-causing microbes, or by the microbes themselves. Antimicrobial susceptibility data shows an alarming increase in the frequency of antimicrobial resistance of foodborne pathogens, a situation which is worrisome as it decreases the effectiveness of drugs employed to reduce the morbidity and mortality associated with serious and life-threatening infections and thus, compromising human health. This study was therefore designed to assess the occurrence and characterization of bacterial foodborne pathogens in various foods sold in Alice, Eastern Cape Province of South Africa in an effort to throw more light on the inherent risk associated with such foods. The study was conducted during the period of 2011 - 2013. Two university restaurants and eight ready-to-eat food vending sites in Alice Town were selected based on their prominence to the students, workers and rest of the community. Microbiological analysis was conducted on 252 samples which included vegetables, potatoes, rice, pies, beef and chicken stew. The isolates were identified using biochemical tests and confirmation of the two most prevalent organisms (Listeria ivanovii and Enterobacter cloacae) was done using PCR techniques. The antimicrobial susceptibility profile of Listeria ivanovii and Enterobacter cloacae strains were identified using the disc diffusion technique; minimum inhibitory concentration was determined by the broth dilution method and M.I.C. Evaluator test strips. The microtiter plate adherence assay was employed to ascertain the ability of these isolates to adhere to a surface whereas the role of cell surface properties in biofilm formation was assessed using the coaggregation and autoaggregation assays. The architecture of the formed biofilms was examined under the scanning electron microscope. The virulence and resistant genes were also detected and characterised by sequencing the PCR products. Bacterial growth was present in all the food types tested; organisms isolated included: Listeria spp. (22%), Enterobacter spp. (18%), Aeromonas hydrophila (12%), Klebsiella oxytoca (8%), Proteus mirabilis (6.3%), Staphylococcus aureus (3.2%) and Pseudomonas luteola (2.4%). PCR confirmed 30 (97%) isolates as E. cloacae complex while 44% (22/50) tested positive for L. ivanovii. All the strains of E. cloacae (100%) and 96% of L. ivanovii isolates (based on phenotypic identification) were resistant to at least four or more of the antibiotics. In this study, bla-TEM was also detected from 48 (96%) of L. ivanovii and 30 (100%) of E. cloacae strains; further analysis of the bla-TEM demonstrated the occurrence of bla-TEM-1. Of the 56 bla-TEM-1 positive isolates sequenced, 7% (4/56) had mutation of either insertion or substitution of a nucleotide. Two virulence genes (ucaA and hlyA) were detected in E. cloacae isolates and none in L. ivanovii using PCR. Sequence analysis of the hsp60 gene reported the presence of two sub-species for E. cloacae; E. cloacae cluster III (75%) and E. cloacae cluster IV (25%); while analysis of the iap60 gene demonstrated that 55.8% (19/34) were L. ivanovii, 44% (15/34) L. seeligeri and 14.7% (5/34) L. welshemeri. A total of 90% L. ivanovii and 88% E. cloacae strains demonstrated the ability to form biofilms; the coaggregation index ranged from 12 to 77% while the autoaggregation index varied from 11 to 55% for L. ivanovii and 27% to 98% for E. cloacae. The findings of this study indicate that most of the ready-to-eat food samples examined did not meet bacteriological quality standards, thus posing potential risks to consumers. This should draw the attention of the relevant authorities to certify that hygienic standards are improved to curtain foodborne infections. Furthermore, the presence of multi-resistant strains is of major concern as these foods could serve as important vehicles transmitting multi-resistant bacteria and genes to humans. In addition the ability of these pathogens to form biofilms may lead to adherence of these organisms to kitchen utensils and other environments leading to cross-contamination of food processed in these areas and increase resistance of organisms to antimicrobial agents.

M.Sc. (Biochemistry) / Cholera, the highly epidemic diarrhoeal disease caused by Vibrio cholerae (V. cholerae) infection, continues to devastate many developing countries. Therefore, a rapid and sensitive method to identify pathogenic V. cholerae biotypes is imperative. Literature highlighted the sensitivity of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and high resolution melt curve analysis (HRM) for this purpose. It is necessary to develop these methods based on the virulence proteins/genes themselves in order to be future diagnostic tools capable of detecting distinct strains of V. cholerae and to determine their pathogenicity. Such virulence proteins/genes would require significant sequence variation among different strains of V. cholerae. The toxin-coregulated pilus protein A (TcpA) is a key player in V. cholerae infection and displays significant sequence variation at both the genetic and protein level. The hypothesis of this study is that the variation in the TcpA/tcpA protein and gene, respectively, can be used to differentiate between V. cholerae biotypes by using MALDI-TOF MS and/or HRM methods. The objectives were first to investigate established methods for the rapid isolation of surface proteins that would yield a high enough concentration of whole pili or TcpA subunits for future MS biotyping techniques. Two methods were evaluated: a) pili isolation by mechanical shearing b) crude extraction of the outer membrane proteins and associated surface proteins. Secondly, HRM-compatible oligonucleotides were designed for specific amplification of the variable regions within the tcpA gene in V. cholerae. The efficacy of these oligonucleotides was tested on V. cholerae reference strains and environmental isolates. Lastly, a current standard procedure for V. cholerae typing was evaluated. Pili were successfully isolated from bacterial cell colonies, but large quantities of starting material were required and much of the cell content was isolated with the pili. Alternative isolation and enrichment methods have been proposed and may prove promising for future MALDI-TOF MS biotyping. The current standard procedure for V. cholerae typing with multiplex PCR revealed some discrepancies between the multiplex PCR steps. Therefore, the current multiplex PCR procedure may result in inaccuracies during typing. The designed HRM-compatible oligonucleotide set designated Contig 1 successfully amplified a 101 bp region within the tcpA gene in Vibrios. This Contig 1 oligonucleotide set could possibly be used in future studies to differentiate between Vibrio species with HRM. Further studies would be needed for the development of MALDI-TOF/MS or HRM as future rapid and specific environmental monitoring systems based on the TcpA/tcpA protein or gene, respectively.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ying Fang / Porcine reproductive and respiratory syndrome virus (PRRSV) causes a tremendous economic loss in swine industry worldwide. The capabilities to evade host immune responses and to establish persistent infection are the two hallmark features of PRRSV infection. In this dissertation, the research was mainly focused on investigating the novel mechanisms underlying PRRSV transmission and persistence. In chapter 2, the research was focused on an alternative pathway of PRRSV intercellular transmission. Our data showed that intercellular nanotube connections can be utilized for cell-to-cell spreading the core infectious viral machinery (viral RNA, certain replicases and structural proteins) of PRRSV. Live-cell movies tracked the intercellular transport of a recombinant PRRSV that expressed green fluorescent protein (GFP)-tagged nsp2 in a receptor-independent manner. The cytoskeleton proteins F-actin and myosin-IIA were identified as co-precipitates with PRRSV nanotube associated proteins. Drugs inhibiting actin polymerization or myosin-IIA activation prevented nanotube formations and viral clusters in virus-infected cells. These data lead us to propose that PRRSV utilizes the host cell cytoskeletal machinery inside nanotubes for efficient cell-to-cell spread. This form of virus transport represents an alternative pathway for virus spread, which is resistant to the host humoral immune response. In chapter 3, we further showed that PRRSV infection could induce the formation of nanotubes between infected and uninfected cells following a ROS-dependent nanotube formation model. Co-culturing PRRSV-infected cells with uninfected cells rescued PRRSV-induced cell death. Mitochondrion was observed transferring from uninfected to PRRSV-infected cells. Importantly, impaired formation of nanotube or defective mitochondrion was unable to rescue infected cells from apoptosis/necrosis. Certain PRRSV proteins were detected to associate with mitochondria and transport from infected to uninfected cells through TNTs. Our results suggest that TNTs-transfer of functional mitochondria rescued PRRSV-infected cells from apoptosis/necrosis in the early stage of infection. On the other hand, mitochondria could be utilized as a cargo to transport viral materials for spreading the infection. In chapter 4, a novel mechanism s of PRRSV persistent infection has been studied. In this study, a cellular model of persistent infection was established. Strand-specific quantitative RT-PCR and RNase I treatment analysis showed that double-stranded RNA (dsRNA) conformation existed in persistently infected cells. This data has been further confirmed in vivo by performing two independent PRRSV persistence studies. Immunohistochemistry analysis showed that viral dsRNAs were detected aggregating inside the germinal centers of tonsils and lymph nodes from PRRSV persistence pigs, but RNA array analysis further showed that dsRNA in lymphoid tissues had limited ability to stimulate host antiviral responses during persistent infection stage. These results suggest that the PRRSV dsRNA functions as a mediator for viral persistence. The viral dsRNA persistence in germinal centers of lymphoid tissues may reveal a novel mechanism for PRRSV to escape antiviral immune responses. In summary, this study investigated two novel pathogenic mechanisms of PRRSV infection, which could provide insights on the development of effective control strategies.