Notes: [Auszug] Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacterium's shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are ...

Notes: We have constructed an aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica, harbouring mutations in aroA and trpE to investigate the use of such a strain as a live-attenuated vaccine. B. bronchiseptica aroA trpE was unable to grow in minimal medium without aromatic supplementation. Compared to the parental wild-type strain, the mutant displayed significantly reduced abilities to invade and survive within the mouse macrophage-like cell line J774A.1 in vitro and in the murine respiratory tract following experimental intranasal infection. Mice vaccinated with B. bronchiseptica aroA trpE displayed significant dose-dependent increases in B. bronchiseptica-specific antibody responses, and exhibited increases in the number of B. bronchiseptica-reactive spleen cells in lymphoproliferation assays. Immunised animals were protected against lung colonisation after challenge with the wild-type parental strain. With such a broad host range displayed by B. bronchiseptica, the attenuated strain constructed in this study may not only be used for the prevention of B. bronchiseptica-associated disease, but also for the potential delivery of heterologous antigen.

Notes: Non-motile mutants of Bordetella bronchiseptica were generated after mini-transposon mutagenesis. One non-motile mutant (designated VMM1) was derived from the bvg-positive strain BB7865 and four mutants (designated AMM1–4) were derived from the isogenic bvg-negative strain BB7866. Southern hybridisation analysis indicated that loss of motility was not due to the disruption of the flagellin subunit gene. Western blot and transmission electron microscopic analysis indicated that three of the five mutants expressed neither the flagellin subunit (40 kDa) nor flagella whereas one mutant expressed intact flagella under all conditions tested. One unique bvg-negative mutant, AMM4, exhibited temperature-dependent repression of flagella biosynthesis and motility at 37°C. The ability of AMM4 to invade and survive in HeLa cells was significantly decreased.

Notes: Bordetella pertussis is the causative agent of whooping cough, a contagious childhood respiratory disease. Increasing public concern over the safety of whole-cell vaccines led to decreased immunisation rates and a subsequent increase in the incidence of the disease. Research into the development of safer, more efficacious, less reactogenic vaccine preparations was concentrated on the production and purification of detoxified B. pertussis virulence factors. These virulence factors include adhesins such as filamentous haemagglutinin, fimbriae and pertactin, which allow B. pertussis to bind to ciliated epithelial cells in the upper respiratory tract. Once attachment is initiated, toxins produced by the bacterium enable colonisation to proceed by interfering with host clearance mechanisms. B. pertussis co-ordinately regulates the expression of virulence factors via the Bordetella virulence gene (bvg) locus, which encodes a response regulator responsible for signal-mediated activation and repression. This strict regulation mechanism allows the bacterium to express different gene subsets in different environmental niches within the host, according to the stage of disease progression.

Notes: Bordetella pertussis is the causative agent of whooping cough. Traditional vaccines against this disease are inherently reactogenic, thus research is currently focussed on the production of less reactive, acellular vaccines. Expression of candidate antigens for these vaccines in Escherichia coli would be preferable, however, several B. pertussis antigens undergo incorrect post-translational processing in E. coli. The leader peptidase gene (lep) of B. pertussis encodes a protein of 294 amino acid residues that shares homology with other prokaryote leader peptidase I sequences. Hydrophilicity analysis based on the predicted amino acid sequence has demonstrated a similar membrane topology to that of E. coli and Salmonella typhimurium leader peptidase I. Co-expression of the B. pertussis lep gene in E. coli strain TOPP2 expressing the pertussis toxin S1 subunit was found to markedly increase the expression and post-translational processing of the S1 protein.

Notes: To investigate the role played by urease during the Bordetella bronchiseptica infection process, the ability to colonise and persist in the mouse respiratory tract of a urease-negative B. bronchiseptica BB7865 and a BB7865 derivative constitutively expressing urease was compared with that of the wild-type strain. The results obtained showed that neither constitutive expression nor abolishment of urease activity had any significant effect on the course of B. bronchiseptica infection. Therefore, under our experimental conditions, urease is not essential for B. bronchiseptica to colonise and persist within the murine host.

Notes: The New York State Water Resources Institute at Cornell University undertook a two-year sampling survey of pesticides in ground water beginning in 1985. The survey focused on areas where combinations of agricultural pesticide use, soil texture, and ground water occurrence seemed likely to lead to leaching. The sampling survey included samples from four types of sampling points: (1) monitoring wells; (2) existing water supply wells; (3) test holes; and (4) tile drains. The monitoring wells were sampled repeatedly throughout the project to attempt to characterize temporal changes in water quality corresponding with seasonal changes in ground water levels. The pesticides studied for this project were atrazine, alachlor, cyanazine, metolachlor, carbaryl, carbofuran (and a metabolite, 3-hydroxy carbofuran), and simazine. All, except for carbaryl, have been found in ground water in other sampling surveys in the United States.The results of the sampling survey did not reflect the careful choices of enviromental characteristics and pesticide use that seemed likely to lead to leaching. Residues of three pesticides were detected in six single samples from separate sources at four of the 30 sites tested. Three of the six samples came from shallow test holes that were used to sample the shallowest possible saturated soils beneath fields. The three pesticides detected were atrazine, simazine, and 3-hydroxy carbofuran. Of the six samples, a single sample from a test hole contained atrazine concentrations equal to the current federal health advisory for long-term exposure to atrazine (3 ppb).The remaining detections were between the limit of detection for analytical methods and the federal health advisory for each pesticide. The federal health advisories were formulated after the end of the project. Analytical methods may have been insensitive with respect to these advisories. Sampling results from other surveys suggest that many detections of the same pesticides lie below the limits of detection used for this sampling survey.A possible explanation for the lack of detections, given the design of the sampling survey, may lie in the agricultural practices noted at sampled sites. Most of the farm managers rotated their crops and pesticides on many small fields. Although the environmental conditions chosen for sampling sites were expected to lead to contamination, reported pesticide applications varied from year to year and field to field according to rotational schedules. The inconsistency of applications from year to year may explain the lack of detections (at the limits of quantification used for analyses) noted in this sampling survey.

Notes: Abstract DNA fragments containing theKlebsiella oxytoca genes encoding β-glucosidase and amylase were cloned into the kanamycin resistance transposon Tn5. Another DNA fragment containing two genes for polygalacturonatetrans-eliminase was cloned into Tn1721. These newly constructed transposons were then each transposed in vivo onto the broad-host-range plasmid pR751 and conjugally transferred to a variety of Gram-negative bacteria. These were then screened for the newly acquired phenotypes.