In order to investigate the role of the ferric uptake regulator Fur in the porcine lung pathogen Actinobacillus pleuropneumoniae, we constructed an isogenic in-frame deletion mutant, A. ...pleuropneumoniae Deltafur. This mutant showed constitutive expression of transferrin-binding proteins, growth deficiencies in vitro, and reduced virulence in an aerosol infection model.
We have investigated the use of a natural transformation protocol to introduce mutations into
Actinobacillus pleuropneumoniae serotypes 1 and 5b. For both strains tested, we recovered 1 in 10
8 ...transformants during culture in rich medium, a result that was independent of the growth phase. This low frequency of transformation of
A. pleuropneumoniae did not increase when bacteria were grown under conditions known to be optimal for induction of competence in
Haemophilus influenzae. Using linearised plasmid DNA containing a kanamycin cassette inserted into the
sodC gene of
A. pleuropneumoniae serotype 1, we showed that natural transformation can be used as a simple method for introducing allele replacements into this bacterium, and can be used to transfer mutations from one serotype to another.
Current porcine pleuropneumonia bacterins afford only partial protection by decreasing mortality but not morbidity. In order to better understand the type(s) of immune response associated with ...protection, antibody- and cell-mediated immune responses (CMIR) were compared for piglets before and after administration of a commercial bacterin, which confers partial protection, or a low-dose (10(5) CFU/ml) aerosol challenge with Actinobacillus pleuropneumoniae CM5 (LD), which induces complete protection. Control groups received phosphate-buffered saline or adjuvant. Serum antibody response, antibody avidity, delayed-type hypersensitivity (DTH), and lymphocyte blastogenic responses were measured and compared among treatment groups to the lipopolysaccharide (LPS), capsular polysaccharide (CPS), hemolysin (HLY), and outer membrane proteins (OMP) of A. pleuropneumoniae. Peripheral blood lymphocytes and sera were collected prior to and following primary and secondary immunization-infection and high-dose A. pleuropneumoniae CM5 (10(7) CFU/ml) aerosol challenge. Serum antibody and DTH, particularly that to HLY, differed significantly between treatment groups, and increases were associated with protection. LD-infected piglets had higher antibody responses (P less than or equal to 0.01) and antibody avidity (P less than or equal to 0.10) than bacterin-vaccinated and control groups. Anti-HLY antibodies were consistently associated with protection, whereas anti-LPS and anti-CPS antibodies were not. LD-infected animals had higher DTH responses, particularly to HLY, than bacterin-vaccinated pigs (P less than or equal to 0.03). The LD-infected group maintained consistent blastogenic responses to HLY, LPS, CPS, and OMP over the course of infection, unlike the bacterin-vaccinated and control animals. These data suggest that the immune responses induced by a commercial bacterin are very different from those induced by LD aerosol infection and that current bacterins may be modified.
Signature-tagged mutagenesis (STM) provided the first widely applicable high-throughput method for detecting conditionally essential genes in bacteria by using negative selection to screen large ...pools of transposon (Tn) mutants. STM requires no prior knowledge of the bacterium's genome sequence, and has been used to study a large number of Gram-positive and Gram-negative species, greatly expanding the repertoires of known virulence factors for these organisms. Originally, hybridization of radiolabelled probes to colony or dot blots was used to detect differences in populations of tagged mutants before and after growth under a selective condition. Modifications of the tag detection method involving polymerase chain reaction (PCR) amplification and visualisation by gel electrophoresis have been developed and can be automated through the use of robotics. Genetic footprinting is another negative selection technique that uses PCR amplification to detect loss of mutants from a pool. Unlike PCR-STM, this technique allows direct amplification of Tn-flanking sequences. However, it requires the bacterium's whole genome sequence in order to design specific primers for every gene of interest. More recently, a number of techniques have been described that combine the negative-selection principle of STM and genetic footprinting with the genome-wide screening power of DNA microarrays. These techniques, although also requiring whole genome sequences, use either a form of linker-mediated or semi-random PCR to amplify and label Tn-flanking regions for hybridization to microarrays. The superior sensitivity microarray detection allows greater numbers of mutants to be screened per pool, as well as determination of the coverage/distribution of insertions in the library prior to screening, two significant advantages over STM.
Does Your Graphing Software Real-ly Work? Marchand, R. J.; McDevitt, T. J.; Bossé, Michael J. ...
PRIMUS : problems, resources, and issues in mathematics undergraduate studies,
20/5/2/, Volume:
17, Issue:
2
Journal Article
Peer reviewed
Many popular mathematical software products including Maple, Mathematica, Derive, Mathcad, Matlab, and some of the TI calculators produce incorrect graphs because they use complex arithmetic instead ...of real arithmetic. This article expounds on this issue, provides possible remedies for instructors to share with their students, and demonstrates that using complex arithmetic has some important advantages.
Supplemental perioperative oxygen (SPO) therapy has been proposed as one approach for reducing the risk of surgical site infection (SSI). Current data are mixed regarding efficacy in decreasing SSI ...rates and hospital inpatient stays in general and few data exist for orthopaedic trauma patients. This study is a phase III, double-blind, prospective randomized clinical trial with a primary goal of assessing the efficacy of 2 different concentrations of perioperative oxygen in the prevention of SSIs in adults with tibial plateau, pilon (tibial plafond), or calcaneus fractures at higher risk of infection and definitively treated with plate and screw fixation. Patients are block randomized (within center) in a 1:1 ratio to either treatment group (FiO2 80%) or control group (FiO2 30%) and stratified by each study injury location. Secondary objectives of the study are to compare species and antibacterial sensitivities of the bacteria in patients who develop SSIs, to validate a previously developed risk prediction model for the development of SSI after fracture surgery, and to measure and compare resource utilization and cost associated with SSI in the 2 study groups. SPO is a low cost and readily available resource that could be easily disseminated to trauma centers across the country and the world if proved to be effective.
We have recently reported the creation of the first immortalized cell line derived from human dermal microvascular endothelial cells (HMEC-1). In preliminary studies this line was found to closely ...resemble microvascular endothelial cells in regard to many phenotypic characteristics. Because two key functional features of endothelial cells are their ability to bind to peripheral blood leukocytes and extracellular matrix proteins via cell adhesion molecules, we have now characterized HMEC-1 in terms of expression and regulation of cell adhesion molecules of the integrin, immunoglobulin gene superfamily, and selectin families. HMEC-1 can either constitutively express or can be induced to express key integrins, including α-1, -2, -3, -4, -5, -6, and -V, as well as β-1, -3, -4, and -5. They also express or are capable of expressing immunoglobulin gene superfamily molecules, such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and a member of the selectin family, E-selectin. A number of important cell adhesion molecules that are either constitutively expressed or that must be induced are regulated in a time- and dose-dependent fashion by selected cytokines. Experiments comparing the phenotypic characteristics of HMEC-1 with human dermal microvascular endothelial cells or human umbilical vein endothelial cells reveal HMEC- 1 to have features of both small- and large-vessel endothelial cells.
The contribution of urease activity to the pathogenesis of Actinobacillus pleuropneumoniae was investigated using 2 different urease-negative transposon mutants of the virulent serotype 1 strain, CM5 ...Nalr. One mutant, cbiK::Tn10, is deficient in the uptake of nickel, a cofactor required for urease activity. The other mutant, ureG::Tn10, is unable to produce active urease due to mutation of the urease accessory gene, ureG. In aerosol challenge experiments, pigs developed acute pleuropneumonia following exposure to high doses (10(6) cfu/mL) of the parental strain, CM5 Nalr, and to the cbiK::Tn10 mutant. When low dose (10(3) cfu/mL) challenges were used, neither urease-negative mutant was able to establish infection, whereas the parental strain was able to colonize and cause lesions consistent with acute pleuropneumonia in 8 of the 20 pigs challenged. These findings suggest that urease activity may be needed for A. pleuropneumoniae to establish infection in the respiratory tract of pigs.
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