Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 110-127). Also available in electronic version. Access restricted to campus users.

Despite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live recombinant Salmonella oral vaccines. Here, a series of recombinant Salmonella Typhimurium strains were constructed to express either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. To investigate different delivery and expression methods, antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in the cytoplasm, or the outer membrane. In addition, combinations of two expression strategies were employed to evaluate the efficacy of combined delivery approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities, the immunogenicity of the constructed recombinant Salmonella strains was evaluated and compared in mice. Using soluble model antigen EGFP, my results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, while eukaryotic expression or colonization with good construct stability is critical for T cell responses. For insoluble antigen model HA, the outer membrane strategy induced better B cell and T cell responses than cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited as initially expected. Based on the advantages, deleterious or synergistic effects of different strategies identified in this study, I conclude that different construction strategies of recombinant Salmonella vaccine strains are needed for different forms of antigens (soluble or insoluble antigens). If the antigen (such as EGFP) is soluble and easily expressed in Salmonella, a low-copy plasmid-based strategy should be employed, as it can provoke both strong B cell and T cell responses with better plasmid stability. If a T cell response is preferred, a eukaryotic plasmid, or chromosome-based, cytoplasmic-expression strategy may achieve better results. For heterologous antigens that are likely to be expressed in an insoluble form inside Salmonella (such as HA), an outer membrane-targeting approach is recommended. In addition, I found that the combination of two expression strategies did not enhance the immune response, and hence I caution the use of such an approach. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Salmonella enterica is a foodborne pathogen of major significance, and as such it has been extensively studied by researchers around the world. However, despite the numerous scientific publications on Salmonella, there are still many gaps in our understanding of its biology. One such gap is in the bacteria's interactions with invertebrate hosts, and in particular, oysters. Nearly 70 million pounds of oysters are consumed in the United States each year, and previous work in the Joens' laboratory found Salmonella in roughly 7% of the market oysters they sampled, with the majority of the isolates being the Newport serovar. The majority of oysters are consumed raw, which makes the presence of Salmonella within oysters a potentially significant food safety problem.To more closely examine the interactions between Salmonella and oysters, the Present Study developed a method to consistently and reproducibly raise oysters in a controlled laboratory environment in order to systematically expose them to enteric bacteria and quantify the amount of surviving bacteria at various time points after the initial exposure. Use of this model system throughout the Present Study led to four main conclusions.The first is that Salmonella enterica serovar Newport is capable of surviving in oysters for at least 60 days, from an average concentration of 3.7x103 CFU/g of oyster meat after 10 days, to over 102 CFU/g of oyster meat after 60 days. The second main conclusion is that the Newport serovar of Salmonella, which was found in such predominance in the earlier Joens' laboratory study, does not appear to have any special adaptations for survival within oysters, as other strains of Newport and other serovars of Salmonella survived equally well within our model. The third main conclusion, based on the results of immunohistochemistry, is that the relationship between Salmonella and oysters is not a transient interaction that is limited to the outside of the oyster's gut epithelium, but involves a long-term colonization inside the oysters' connective tissues. Because the survival of Salmonella in oysters could be of a pathogenic nature, the Present Study knocked out two key type III secretion systems (T3SS) found in two distinct Salmonella pathogenicity islands (SPI-1 and SPI-2) known to be critical for pathogenesis in mammalian hosts and examined their role in the bacteria's ability to survive within oysters. The results revealed that neither the SPI-1 nor the SPI-2 T3SS were necessary for Salmonella's survival in oysters, which led to the final conclusion of the Present Study that the nature of Salmonella's infection of oysters is fundamentally different than the pathogenesis that occurs in mammalian hosts and that further study of the mechanisms of the survival of Salmonella in oysters is needed to better understand the important and interesting relationship between a significant source of food and this common, and occasionally deadly, foodborne pathogen.

Despite their beneficial effects, concerns have been raised about the role of antimicrobial growth promoters (AGP) in the emergence of antibiotic resistant bacteria. This study evaluated the effects of approved AGP on the emergence of antibiotic resistance in commensal E. coli and foodborne pathogen Salmonella. A survey of antibiotic resistance levels in commercial broiler chicken farms in the Fraser Valley (B.C.) and an experimental feeding trial were conducted from May 2004 to February 2005 and May to November 2005, respectively. The latter examined the effects of ten AGP formulations (bambermycin, penicillin, salinomycin, bacitracin, combination of salinomycin and bacitracin, chlortetracycline, virginiamycin 11ppm, virginiamycin 22ppm, monensin and narasin) on bird performance as well. Multiple antibiotic resistant commensal E. coli and Salmonella carrying virulence genes were found at commercial broiler chicken farms and therefore may serve as reservoirs for these genes. There was no significant difference between feed formulations on the phenotypic or genotypic characteristics of the isolates, except for tetracycline resistance gene tet(B). In the experimental feeding trial, broiler chickens were fed a diet including or excluding AGP. Birds were sampled prior to and weekly during feeding of the control and the AGPP containing diets. Although not detected on day 0, E. coli increased after day 7 to more than 9.9 log10 CFU/g in ceca. Multi-drug resistant E. coli were isolated from birds fed the ten AGP containing diets as well as the control diet. Except for penicillin, none of the AGP containing diets significantly improved bird performance compared to the control diet (P>0.05). Good management practices can significantly improve broiler chickens performance and decrease the mortality rate.

Tectiviridae šeimos bakteriofagas PRD1, infekuojantis dauginiu atsparumu antibiotikams pasižyminčias gramneigiamąsias bakterijas, patekimui į ląstelę naudoja viriono viduje esančią membraną. Manoma, kad šio bakterijų viruso membrana patekimo metu suliejama su infekuojamų ląstelių plazmine membrana. Deja, tiesiogiai įrodyti, kad faginė membrana susilieja su infekuojamų bakterijų plazmine membrana yra sudėtinga. Registruodami fago sukeliamas TPP+ ir K+ sroves bei tirdami membranose aktyvių antibiotikų poveikį membranos įtampai ir kalio jonų gradientui pabandėme ištirti Salmonella enterica ląstelių plazminės ir išorinės membranų laidumo pokyčius pradiniame infekcijos etape ir nustatyti infekcijos terpės fizikocheminių savybių įtaką bakteriofago PRD1 sąveikai su S. enterica ląstelėmis. Bakteriofagas PRD1, infekuodamas Salmonella enterica ląsteles, didina išorinės membranos laidumą, bet nedepoliarizuoja jų plazminės membranos. 300 OsmM ir didesnis infekavimo terpės osmosinis slėgis bei 22 oC ir žemesnė temperatūra slopina bakteriofago PRD1 poveikį Salmonella enterica ląstelių apvalkalėlio laidumui. Skirtingai nuo giminingo bakteriofago Bam35 infekcijos poreikių, dvivalenčiai katijonai nėra reikalingi fago PRD1 sąveikai su S. enterica ląstelių apvalkalėliu pradiniuose infekcijos etapuose. Dvivalenčių katijonų kompleksonai net skatina membranas laidinantį fago PRD1 poveikį, o Ca2+ ir Mg2+ jonai šiek tiek slopina plazminės membranos laidinimą. Arsenatas slopina PRD1 poveikį... [toliau žr. visą tekstą] / Bacteriophage PRD1 from Tectiviridae family uses its internal membrane to enter the host multiple resistant to antibiotics Gram-negative bacteria. Some indirect evidences indicate that during the entry phage membrane fuses with the plasma membrane of bacterial cell. However, it is very complicated to get the direct evidences or indications of the membrane fusion in this system. Monitoring the phage-induced fluxes of TPP+ and K+ ions and studying the influence of membrane-active antibiotics on membrane voltage and potassium gradient we were investigating PRD1-induced changes in the permeability of the outer and the plasma membranes of Salmonella enterica cells and registering the influence of physicochemical parameters of infection medium on the efficiency of phage-cell interaction. We have shown that phage PRD1 increases permeability of the outer membrane but does not depolarize the plasma membrane during the entry. 300 OsmM and higher osmotic pressure in the infection medium and temperature lower than 22 oC blocks the phage induced changes in bacterial envelope. Differently from the relative bacteriophage Bam35, divalent cations are not needed for PRD1 entry. Complexones of divalent cations EDTA and EGTA stimulate, but Ca2+ ir Mg2+ slightly inhibit the plasma membrane-permeabilizing activity of PRD1. Arsenate inhibits PRD1 induced changes of the cell envelope and the reduced intracellular ATP content could be the reason. Bacteriophage-induced lysis of S. enterica cells is... [to full text]

After two outbreaks of salmonellosis that were linked to the consumption of almonds in 2001 and 2004, scientific community and industry have encouraged the study of pathogen inactivation kinetics in almonds. However, these studies often have overlooked the increase of water resulting from the inoculation of almonds. This increase of free water may result in data that may be overestimating the final outcome of treatments applied to pasteurize almonds in industrial settings. Therefore, in 2011 a study of almonds was performed at Texas A&M University to determine whether there was a need for Aw restoration. In this study, the effect of accelerated drying on the reduction of Salmonella on almonds was investigated, Salmonella Entritidis PT 30 isolated from one of the outbreaks linked to almonds, and Salmonella Typhimurium LT2 were used. For irradiation, the effect of vacuum packaging and air packaging on the sensitivity of Salmonella to e- beam irradiation was evaluated for both accelerated drying and conventional drying. The D value for Salmonella Entritidis PT 30 when treated with blanching at 88°C with accelerated drying was 10.7± 0.1 s, and 12.8 ± 0.3 s with conventional drying. When subjected to oil roasting at 127˚C, the D values were 10.5 ± 0.1 s with accelerated drying, and 10.2 ± 0.2 s with conventional drying. For irradiation treatments, the D10 value for vacuum packaged almonds was 0.35 ± 0.02 kGy with accelerated drying, and 0.38 ± 0.04 kGy with conventional drying. When irradiated in the presence of air, the D10 value of Salmonella was 0.26±0.04 kGy for almonds with accelerated drying, and 0.29 ± 0.03 kGy conventional drying. In conclusion, the accelerated drying process resulted in greater reduction in Salmonella on almonds in comparison to conventional drying when subjected to blanching but no differences were found for oil roasting (P > 0.05). For e-beam irradiation the D_10 values were significantly greater (P < 0.05) for vacuum with accelerated drying than for Air almonds with accelerated drying. This indicates that if a process applied in the industry were to be developed based on challenge studies when conventional drying was achieved, the almonds might likely be subjected to a treatment that is stronger than necessary to achieve pasteurization.

RdoA is a eukaryotic-like serine/threonine protein kinase found in Salmonella typhimurium. It is a downstream effector of the Cpx stress response pathway and has been phenotypically characterized to have a functional role in flagellin phase variation and long-term bacterial survivability. Structurally, RdoA is homologous to, choline kinase and aminoglycoside (3’) phosphotransferase IIIa (APH[3’]IIIa). These kinases all belong to a protein kinase superfamily and share highly conserved residues/motifs in their catalytic domain. In RdoA seven of these conserved amino acids were proposed to have functional roles in the phosphotransfer mechanism. Mutation of these proposed catalytic domain residues resulted in a loss of in vitro kinase activity and in vivo RdoA function for a majority of the mutants. Four of the mutants also exhibited decreased levels of stable RdoA compared to wildtype. Many protein kinases regulate activity through phosphorylation of an activation loop. Although RdoA does not contain a canonical activation loop, its carboxyl terminus is proposed to play a similar regulatory function. Mutations of a putative autophosphorylation target in the carboxyl terminus resulted in loss of in vitro kinase activity. Truncations of this region also resulted in loss of kinase activity, as well as decreasing RdoA stability. The length of the carboxyl terminus in the kinase was shown to be an important determinant in the overall structural stability of RdoA. Mutational analyses of conserved amino acid residues surrounding the putative substrate-binding cleft of RdoA revealed site specific mutants with diminished in vitro phosphorylation activity and/or RdoA levels. A subset of these mutants for which no in vitro kinase activity was detected were still able to complement RdoA function in vivo. Taken together these results indicate that this region of the protein is important for RdoA function. In summary, this work has generated a panel of RdoA mutants with several unique phenotypes that will facilitate characterization of RdoA function and of regions of the protein / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2010-09-29 21:35:42.815

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Four experiments were conducted to evaluate effects of arginine (ARG), vitamin E (VE) and mannanoligosaccharide (MOS) on immune response and clearance of Salmonella in broiler chickens. There were 4 groups in Exp.1 (E.1) and E.2: antibiotic-free diet (CTL-); diet with antibiotic (CTL+); ARG and VE (AVE); ARG, VE and MOS (AVM). Birds were infected with 10^6 CFU/ml of a novobiocin and nalidixic acid resistant Salmonella enteric Serovar Typhimurium (ST) at d 7 (E.1) or at d 3 (E.2). In E.3 and E.4, there were three groups: CTL, AVE and AVM; at d 3, birds in E.3 were infected with 10^2 CFU/ml ST and birds in E.4 were infected with 10^6 CFU/ml ST. The following were analyzed for four experiments: Oxidative burst heterophils response (OBHR) and monocytes response (OBMR); Lymphocyte proliferative response (LP); antibodies concentrations in serum; chicken body weight (BW), cecal Salmonella concentration; gut parameters. In E.1, both AVM and AVE diet decreased OBHR; birds fed AVM showed the highest IgA level, birds fed AVM had higher IgM than CTL+ and had the lowest Salmonella population. Birds fed AVE showed higher LP than CTL- in E.1 and E.2. From E.1 and E.2, AVM decreased chicken innate immune response at a younger age and improved adaptive immune response when birds were older than 16 d. AVM reduced ST when administrated later (d 7) versus early (d 3). In E.3, no BW and Salmonella counts differences were found; birds fed AVM had a higher IgA than AVE but similar to CTL at d 17. Chickens fed CTL had the highest OBHR at d 17, chickens fed AVM had the highest LPR at d 17. In E.4, chickens fed AVM showed higher BW than CTL at d 17 and d 24. Also no Salmonella counts differences were found. Chickens fed AVE had higher LPR than chickens fed AVM at d 17. These results are consistent with results from E.1 and E.2. Overall, diet AVM has different effects on chicken immune response when chicks were infected at different age; higher level of ARG and VE improved immune response and improve gut health.