2006 merck/merial - School of Veterinary Medicine - Louisiana State ...

isolated to analyze cytokine gene expression. The in-vivo model involves infecting day-old chicks with C. jejuni andeuthanizing within 24 hours of infection in order to monitor the acute response of the gastro-intestinal epithelium to thebacteria. Cecal, ileal and kidney samples were collected for quantitative RT-PCR analysis of expression of variousinflammatory cytokines, transmission electron microscopy and bacterial enumeration. Cytokines monitored for this studyinclude chicken IL-8, IL-6, IL-1b and b-D2. These analyses are ongoing. We anticipate no significant up-regulation of theselected inflammatory cytokines in both the in-vitro and in-vivo models, confirming the failure of the chicken to mount anacute immune response when exposed to C. jejuni. If our results verify our hypothesis, the in-vitro studies would bevalidated as a model system to study the nature of this commensal relationship. The long term aim of this work is todetermine the mechanism by which C. jejuni is able to colonize chickens without causing significant symptoms. Supported inpart by the Merck-Merial Veterinary Scholars Grant to Michigan State University and a grant from the USDA Food SafetyProgram to VJD.A Comparison of Interaction Patterns of a Wild Type Salmonella Enteritidis Strain and its Three Mutantsin Experimentally Infected Egg-Laying HensMaley, Jessica R.*, Crisp, Nicole S., Cho, Seongbeom, and Saeed, A. MahdiMichigan State University College of Veterinary MedicineNational Food Safety and Toxicology Center at Michigan State UniversityProgram in Infectious Diseases and Immunity at University of California at BerkeleySalmonella Enteritidis is becoming an increasingly common cause of foodborne illness. To understand the virulenceprofile of S. Enteritidis, three mutant strains (M1, M2, and M3) were generated from a wild type (WT) S. Enteritidis, phagetype 4, using transposon-mediated mutagenesis. By analyzing the characteristics of these mutant strains and comparing theirin vivo interactions to that of their wild type parent strain, we hope to further understand the impact of the altered genes.Forty egg-laying hens were confirmed as baseline negative for S. Enteritidis by bacteriological fecal and egg culture andrandomly separated into four groups of ten. Nine hens in each group were inoculated with 1 X 109 CFU of their respectivestrain, and then monitored for twenty-eight days for colonization of internal organs, fecal shedding of S. Enteritidis, and theproduction of S. Enteritidis contaminated eggs. Select positive samples were further evaluated using Multilocus VariableTandem Repeat Analysis (MLVA) and in vitro attachment and invasion assays using Hep-2 cells. S. Enteritidis was morereadily isolated from feces than eggs. In hens inoculated with WT and M1 strains, the rate of isolation of SE from organs andfeces was greatest initially and tapered off as the experiment progressed. Hens inoculated with M2 and M3 deviated fromthis trend, as fairly high concentrations were present later in the experiment. The rate of S. Enteritidis isolation was highestin the cecum, moderately high in the liver and spleen, and reduced in the ovary. The MLVA profiles for S. Enteritidis strainswere identical before and after inoculation. Random shortening of a specific allele was noted. Attachment and Invasionassays are currently underway. The increased persistence of mutant strains M2 and M3 are likely resultant of advantagesawarded by their mutations. M2, which carries an altered restriction enzyme, may produce proteins affected in such amanner that antigenic identification could be modified, enabling the mutant to evade the host’s defense mechanisms. In asimilar manner, M3, which exhibits an altered metabolic profile, may have modified antigenic properties and/or an enhancedability to survive in diverse physiologic conditions. Supported in part by grant number NIHT-35RR017491 to Michigan StateUniversity.Assessment of vaginal ecosystem diversity by analysis of terminal restriction fragment lengthpolymorphism (T-RFLP) of the 16S rRNA genes.Corrin McCann*, Noriko Nakamura, Mengfei Ho, Angel Rivera, Claudia Reich, H. Rex Gaskins, Lois L. Hoyer,Steven Blanke, James M. Slauch, Gary J. Olsen, Brenda A. WilsonUniversity of Illinois at Urbana-Champaign, Institute for Genomic BiologyHost-Microbe SystemMultiple genera and species of microbial populations colonize areas of the body such as the vagina Little is currentlyknown about the diversity of vaginal ecosystems and changes in populations of microbiota in disease states. Our researchhopes to gain information as to the normal microbiota of the vagina and what role it plays in vaginal health and disease. Theobjective of this study is to examine the diversity of human vaginal microbiota using the analysis method of terminalrestriction fragment length polymorphisms (T-RFLP) of the 16S rRNA genes. With a more complete understanding of therole that normal microbial populations play in vaginal health we hope to identify factors that may predispose certain femalesto acquiring vaginal infections or other STDs. We hypothesized that in disease states certain microbial populations would besignificantly over or under-represented affecting the diversity of the T-RFLP profile. This study will determine the variationin microbial diversity within and between individual subjects as a function of sampling location and method. In order toanalyze the vaginal samples, we first needed to optimize the conditions for T-RFLP analysis. To accomplish this weexamined primer sets (27fbac & 1492r, 27f & 926r), DNA template concentration for PCR amplification, purified PCRproduct DNA concentration, digestion enzymes (Hae III, Hha I, Msp I), and digestion conditions (enzyme concentration andincubation length) for optimal resolution of bacteria with minimal time and supplies. Seven pure cultures were used tooptimize the protocol conditions (Clostridium bifermentans, Clostridium paraputrificum, Enterococcus faecalis, Lactobacillus124