Browsing by Subject "Real-time PCR"
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Item Autoinducer 2-based quorum sensing response of Escherichia coli to sub-therapeutic tetracycline exposure(Texas A&M University, 2006-10-30) Lu, LingengAutoinducer 2 (AI-2) is a quorum sensing signal employed by bacteria to coordinate their response to environmental stresses. The objective of this study was to determine the relationship between presence of AI-2 molecules, exposure to sub-therapeutic tetracycline, the expression of genes associated with the conjugal transfer of antibiotic resistance plasmids, and the conjugal transfer of these plasmids in Escherichia coli. The studies showed that AI-2 activity increased in Tets E. coli in the presence of tetracycline (2 ????g/mL) under both batch and continuous culture conditions. The presence of AI-2 molecules induced tetracycline tolerance development in Tets E. coli. The studies showed that the survival rates of Tets E. coli exposed to AI-2 molecules were significantly higher compared to the cells not exposed to AI-2 molecules or cells that were exposed to only LB (Lauria-Bertani) broth. Molecular analyses using real-time PCR indicate that the expression of at least one conjugation-associated gene (trbC) is increased 9-fold in cells exposed to AI-2 molecules in the presence of sub-therapeutic tetracycline compared to its negative controls. The transconjugation frequency of the plasmid RP4 carrying the tet(A) gene increased between 10-100 fold in the presence of AI-2 molecules. In companion studies, AI-2-like activity was detected in fish, tomatoes, cantaloupes, carrots and milk samples. Interestingly, ground beef and poultry meat contained substances that appear to inhibit AI-2 activity. Collectively, these results highlight the potential importance of bacterial quorum sensing signals such as AI-2 in the response of bacterial cells to environmental stimuli and the possible role of quorum sensing signals in the quality and safety of foods.Item Establishment, identification, quantification of methanogenic archaea in chicken ceca and methanogenesis inhibition in in vitro chicken ceca by using nitrocompounds(Texas A&M University, 2006-08-16) Saengkerdsub, SuwatIn the first phase of this study, the diversity of methanogenic bacteria in avian ceca was found to be minimal. Based on 16S rDNA clone libraries, a common phylotype, designated CH101, ranged between 92.86 to 100 % of the total clones whereas less than 1% of the other phylotypes were found. On the basis of the sequence identity, all of the sequences, except sequence CH1270, are related from 98.97 to 99.45% to 16S rDNA Methanobrevibacter woesei GS. Sequence CH1270 is 97.62% homologous to the sequence identified to uncultured archaeon clone ConP1-11F. Clearly, the predominant methanogen found to reside in the chicken ceca was M. woesei. By using a MPN enumeration method, methanogen counts were found to be in the range of 6.38 to 8.23 log10 organisms per gram wet weight. The 16S rDNA copy number per gram wet weight in the samples was between log10 5.50 and 7.19. The second phase of the study was conducted to observe the effects of selected nitrocompounds and two different feedstuffs on in vitro methane production in chicken cecal contents and rumen fluid. Initially, one of the three nitrocompounds was added to incubations containing cecal contents from laying hens supplemented with either alfalfa or layer feed. Both feed materials influenced volatile fatty acids (VFA) production and also fostered methane production in the incubations although methane was lower (P < 0.05) in incubations with added nitrocompound, particularly nitroethane. Secondly, nitroethane was examined in incubations of bovine or ovine rumen fluid or cecal contents containing either alfalfa or layer feed. Unlike cecal contents, layer feed significantly (P < 0.05) supported in vitro methane production in incubations of both rumen fluids. The results show that nitroethane impedes methane production, especially in incubations of chicken cecal contents. The final phase of this study was carried out to determine the methanogenic establishment in the chicken ceca by the cultural method with the quantitative PCR. The results suggested that methanogens colonized in chicken ceca at a few days after birth. Litter and house flies could be potential sources for methanogenic colonization in broiler chicks.Item Modulation of cell yields and genetic responses of Salmonella fermentation and colonization in the gastrointestinal ecology of avian species(2009-05-15) Dunkley, Kingsley DelroyIn these studies we evaluated specific environmental stimuli relevant to Salmonella virulence and physiology in the gastrointestinal tract of chickens. Results from Salmonella growth in steady state, glucose-limiting continuous culture (CC) indicated that the optimal growth condition was observed between 0.05 h-1 and 0.27 h-1 dilution rates (D). Cell protein concentrations increased proportionally with an increase in D at each steady state, but after D 0.27 h-1 there was a reduction in the cell protein concentrations as the D increased. Genetic responses generally indicated that the lowest D exhibited highest hilA relative expression. Relatively higher expression of hilA was largely observed at low D (low glucose) (0.0125 h-1, 0.025 h-1, 0.05 h-1). Salmonella incubated in CC at different pH shifts demonstrated that cell protein concentration, glucose utilization, Yield ATP and Acetate:Propionate ratios were influenced by an increase in pH (6.14 to 7.41). These parameters increased and decreased consistently with a corresponding increase and decrease in pH. Polymerase chain reaction-based denaturant gradient gel electrophoresis showed that the overall amplicon band patterns of microbial similarity have demonstrated that hens molted with Alfalfa (ALC+) diet were similar to the Full-Fed (FF+) treatment group. Additional, FF+ and ALC+ treatment groups exhibited a higher percentage similarity coefficient (>90%) than the feed deprived treatment group. Fermentation response from cecal inocula on feed substrates revealed that alfalfa based samples yielded consistently higher short chain fatty acid levels when compared to other feed substrates. Salmonella Enteritidis (SE) colonization in liver, spleen and ovaries was significantly (P < 0.05) higher in FW+ hens compared to ALC+ and FF+ treatments groups. A 4-fold (log10 1.29) reduction in SE colonization for ALC+ hens compared to feed withdrawal hens (FW+) (log10 5.12) SE colonization was observed. Relative expression of hilA in all treatment groups was significantly (P < 0.05) higher in FW+ compared to FF+ and ALC+ groups. hilA expression in FW+ hens was 3.2-, 4.2-, and 1.9-fold higher for Days 6, 11 and 12 respectively, when compared with to ALC+ hens. These results suggest that Salmonella virulence in the gastrointestinal ecology of chickens could be impacted by a combination of low nutrients availability and pH shifts.Item New Insights Into the Role of Equine Infectious Anemia Virus S2 Protein in Disease Expression(2011-08-08) Covaleda Salas, Lina M.Equine infectious anemia virus (EIAV) is an important animal model to study the contribution of macrophages in viral persistence during lentiviral infections. EIAV is unique amongst the lentiviruses in that it causes a rapid, rather than the very slow disease progression, characteristic of other lentiviral infections. The accessory gene, S2, unique to EIAV, is an important determinant in viral pathogenesis. A functional S2 gene is required to achieve high-titer viremia and the development of disease in infected horses. Despite its essential role, the mechanisms by which S2 influences EIAV pathogenesis remain elusive. The goal of this research was to gain insight into the role of S2 in pathogenesis. To accomplish this goal we: (i) Examined the effects of EIAV and its S2 protein in the regulation of the cytokine and chemokine responses in macrophages, (ii) Assessed the influence of EIAV infection and the effect of S2 on global gene expression in macrophages and (iii) Identified host cellular proteins that interact with S2 as a starting point for the identification of host factors implicated in S2 function. The results from this study provide evidence for a role of S2 in enhancing a proinflammatory cytokine and chemokine response in infected macrophages. Specifically, S2 enhances the expression of IL-1 alpha, IL-1 beta IL-8, MCP-2, MIP-1 beta, IP-10 and a newly discovered cytokine, IL-34. Involvement of S2 in cytokine and chemokine dysregulation may contribute to disease development by optimizing the host cell environment to promote viral dissemination and replication. Microarray analyses revealed an interesting set of differentially expressed genes upon EIAV infection. Genes affected by EIAV were involved in the immune response, transcription, translation, cell cycle and cell survival. Finally, we used the yeast two-hybrid system to identify S2 host cellular interacting proteins. We identified osteosarcoma amplified 9 (OS-9) and proteasome 26S ATPase subunit 3 (PSMC3) proteins as interacting partners of S2. Additional evidence is needed to demonstrate the physiological relevance of these interactions in vivo. In summary, the results from this study contribute towards our understanding of the role S2 in disease expression and allow the formulation of new hypotheses as to the potential mechanisms of action of S2 during EIAV infection.