No description available.

No description available.

Attempts to produce an effective vaccine against group B meningococcal disease have been unsuccessful due to poor immunogenicity of the capsular polysaccharide, and high variability in the currently identified outer membrane proteins. There is therefore a need to find other vaccine candidates. It is clear from studies of bacteria such as Salmonella typhimurium, that successful pathogens have the ability to specifically express genes in response to an in vivo environment. Many of these in vivo-expressed gene products will contribute to virulence of the organism and may therefore be target antigens for development of new vaccines. An in vivo promoter-probe strategy was developed using the Cre/lox recombination system to identify meningococcal promoters that are specifically expressed in vivo. The strategy employs the cre gene, which encodes a site-specific recombinase that targets two 34-base pair directly repeated lox sites. A vector, pUS1604, was constructed which contains two lox sites flanking a transcriptional terminator that separates the aph (encoding kanamycin resistance) coding sequence from its promoter. A promoterless ere gene was placed on a low copy number vector to limit the number of Cre molecules within each host bacterial cell. Expression of Cre is detected by its ability to recombine the two lox sites and reconstitute a functional kanamycin resistance gene. The system was initially tested in E. coli by cloning a known regulated promoter from the araBAD operon that is induced by L-arabinose and repressed by D-glucose. However, the promoter failed to tightly control the regulation of cre gene expression, resulting in partial loxP recombination, which produced a mixed population of streptomycin and kanamycin resistant cells within each colony. Also, attempts to place the system into the meningococcus were unsuccessful. Efforts were made to tighten the regulation of the cre gene by placing the system into a different E. coli strain and test the system using a library of meningococcal genomic DNA fragments, as a compromise to show the experiments intended to isolate promoters. A vector, pUS1623, was constructed that would place the cre gene under the control of external promoters. A group B meningococccal genomic library was cloned upstream of the promoterless cre gene, with the inserted DNA fragments ranging from 0.1-1kb in size. The library was introduced into the E.coli XLl-Blue strain carrying pUS1604, and the Cre/lox recombination event has been shown to behave as expected, yielding a proportion of kanamycin-resistant clones. This indicates that promoter-like sequences are driving Cre, which is seen through the kanamycin-resistant phenotype of the clone. Conversely, clones that remain streptomycin-resistant do not have promoterlike sequences driving the expression of Cre. This streptomycin-resistant pool of clones was used to inoculate 50% serum/50% PBS liquid medium to identify group B strain meningococcal promoter sequences that drive Cre expression in this environment. Several sequences found to induce Cre expression from the initial screen, and during the serum experiment were sequenced. The sequence data was next analysed for homology against the sequences on the group B meningococcal genome sequence database (http://www.tigr.org). to identify the function of the contig from which the isolated sequence is found within the meningococcal genome. Finally, the sequences were analysed for prokaryote -35 and -10 consensus regions and orientated to determine the direction of transcription relative to the cre gene and also to genomic open reading frames, to determine the likelihood of isolating actual promoters. Although the experiments carried out in E. coli do not give an accurate picture of the regulatory gene pathways in the meningococcus, the results give an insight into the suitability of the system as regards to its use in an IVET strategy.

Infectious diseases continue to be major health concerns worldwide. Although major advances have led to accumulation of genomic data about human pathogens, there clearly exists a gap between genome information and studies aiming at identifying potential drug targets. Here, constraint-based modeling (CBM) was deployed to integrate disparate data types with genome-scale metabolic models (GEMs) to advance our understanding of the pathogenesis of infectious agents with respect to identifying and prioritizing drug targets. Specifically, genome-scale metabolic modeling of multiple stages and species of Plasmodium, the causative agent of malaria, was used to prioritize potential drug targets that could be used to simultaneously treat (anti-malarials) and block transmission of the parasite. In addition, species-specific metabolic models were used to guide translation of findings from non-human experimental disease models to human-infecting species. Further, comparative analysis of the essentiality of metabolic genes for V. cholerae, the causative agent of cholera, growth and survival in single and co-infections with other enteric pathogens led to prioritizing conditionally independent essential genes that would be potential drug targets in both single and co-infection scenarios. Taken together, our findings highlight the utility of using genome-scale metabolic models to prioritize druggable targets that would be of broader spectrum against human pathogens.

The increase in production farming, also known as concentrated animal feeding operations (CAFOs), garners more investigations on the implications to public health regarding the disposal of the wastes of food production animals. In addition to the vast amount of animal manure produced, human biosolids is another waste residual that must be managed. The research focus was the sustainability of foodborne pathogens in waste products and the variables that manipulate these environments such as moisture, temperature, organic matter and time. The first study was designed to analyze spatial differences in microbial populations in broiler litter by investigating the relationship of intra-house location, age of flock, bedding moisture, and seasonality. Antibiogram profiles of selected isolates were explored to determine if antibiotic resistant bacteria are common in these environments and if multiple class resistance is present. These findings provided insight into new targets that may reduce zoonotic bacteria that are problematic from a food safety prospective as well as nuisance bacteria that threaten broiler health. The second study was designed to establish current decay rates of viral and bacterial pathogens when seeded in various waste residuals and the effects soil type and application method have on those rates. Decay rates were established by standard culture and molecular methods, such as qPCR. A comparison of both derived inactivation rates were analyzed to determine if these methods were significantly different. Both cultural and molecular methods have limitation and advantages, and the argument that both are useful and needed is asserted. The decay rates associated with each method were used to simulate a one-time exposure to a land application site to assess the microbial risk of Salmonella using a Quantitative Microbial Risk Assessment model.

No description available.

Aquaculture has potential to support the food supply of increasing world population. Flavobacterial diseases pose a serious problem in wild and aquacultured fish stocks throughout the world. Flavobacterium columnare, F. branchiophilum, and F. psychrophilum are well-known Flavobacterium species that cause important fish losses. Recently, new Flavobacterium species, isolated from diseased fish, have been reported, but their virulence mechanisms are not clear. Thus, the goal of this study was to understand pathogenicity of Flavobacterium species. To this goal, 86 Flavobacterium genomes were analyzed by comparative genomics. Predicted virulence genes were identified for all genomes. For each species, unique and shared virulence genes were determined. For all genomes, unique and common predicted antibiotic resistance genes were identified as well. Secreted proteins are important virulence factors. Thus, all encoded secretion and related systems were determined. By using different genomics approaches, F. columnare genomovar I (highly virulent to cold-water fish species like trout) and genomovar II (extremely virulent to warm-water fish species such as catfish and tilapia) genomes were analyzed, and transposon mutants using Tn4351 in six F. columnare genomovar II strain 94-081 were generated. The hemolysin and glycine cleavage protein mutants had 15% and 10% mortalities, respectively while wild-type strain caused 100% mortality. Potential virulence genes, unique proteins, and other genomic features of F. columnare genomovars were determined. Mutants targeting unique genes in valine-leucine-isoleucine biosynthesis pathway were constructed. The virulence of Fcol(DELTA)leuD and Fcol(DELTA)ilvD mutants exhibited reduced virulence.

No / This paper illustrates the characteristics of plant pathogens that have been found to be of most relevance in offensive biological warfare programmes. It shows how states envisaged these pathogens might be used against crops. It assesses whether the Biological and Toxin Weapons Convention can deal adequately with this potential threat.

Campylobacter fetus, a microaerophilic, Gram-negative rod, is a well-known cause of contagious abortion and infertility in cattle and sheep and is gaining increasing recognition as an opportunistic human pathogen. In the past, the unusual oxygen requirements of the organism have complicated its recovery from clinical sources; optimum recovery necessitates the use of special gas mixtures, vacuum pumps, etc., not routinely used in most laboratories. In this study, the stimulatory effects of compounds found to enhance aerotolerance and growth of C. fetus were tested for 62 strains of C. fetus, representing each subspecies, to test the desirability of supplementing conventional media with these additives. Brucella agar supplemented with 0.025% (each) FeS04·7H20, sodium bisulfite, and pyruvic acid (FBPA agar) supported growth of 82% of the strains tested under simulated candle jar condtions. Brucella broth supplemented with 0.2% FeS04 ·7H20, 0.025% sodium bisulfite, and 0.050% pyruvic acid (FBPB broth) supported growth of 61 of 62 strains at 21% 02, 2.5% CO2 with static incubation. Therefore, FBPA agar and FBPB broth are recommended for the isolation and cultivation of C. fetus. Although isolation from clinical sources is still dictated to some extent by the oxygen tension used for cultivation, improved recovery may be expected regardless of equipment or facilities available. Another compound found to enhance aerotolerance of C. fetus was SOD. This finding supports the hypothesis that the stimulatory effect of the media additives results from a direct action on the culture medium by degrading toxic derivatives of oxygen, such as the superoxide radical and hydrogen peroxide. / Master of Science

Master of Science / Department of Agronomy / DeAnn Presley / In the Midwest there has been an increase in the number of vertical tillage (VT) implements sold and a large push in marketing these newer implements to producers. Vertical tillage is defined as shallow tillage, usually in the top 5 to 7.5 cm of the soil and results in no horizontal disturbance of the soil. The objective was to determine the short-term (one growing season) effects of a vertical-tillage operation on seedling emergence, crop growth and development, yield, residue decomposition, disease incidence and severity, quantification of pathogen propagules in soil and crop residue, and effects on the near-surface soil physical properties. The study was conducted during the 2010 and 2011 growing seasons at nine locations total for the two years throughout Kansas. The study compared vertical tillage against the producer’s current practice of no-till (NT), strip tillage (ST), or conventional disk (CD). Few significant differences were observed when studying soil properties, however not one treatment continuously had significant results and no trend was observed. Residue cover at all sites and across both years was significantly greater in the NT treatments. The residue cover also impacted the disease incidence and severity of Cercospora zea-maydis also known as gray leaf spot (GLS). Other diseases such as Marcophomina phaseolina and Fusarium spp. were not significantly impacted by one treatment or another. Overall, any differences in the soil, plant, and pathogen indicators have not resulted in significant yield improvements at any of the nine site locations of the two years of this study, but more site years will be needed to assess any potential benefits of VT. Information gained from this project will be disseminated to extension clientele including extension educators, producers, commodity groups, and agricultural professionals.