Production and purification of recombinant protective antigen and protective efficacy against Bacillus anthracis.

Recombinant protective antigen (rPA), expressed by Bacillus subtilis WB600 (pPA 101), has been purified to homogeneity and the protective efficacy against a Bacillus anthracis challenge has been investigated. rPA was fractionated from culture supernatant fluid by ammonium sulphate, followed by anion exchange chromatography using DEAE Streamline, anion-exchange chromatography on FPLC MonoQ HR 10/10 and finally, gel filtration chromatography on FPLC Superose 12 HR 10/30, to yield 7 mg rPA per litre of culture. The protective efficacy of rPA against an airborne challenge with the AMES strain of B. anthracis was determined in the presence of the adjuvants, alhydrogel and Ribi, and compared to that achieved by the current UK human vaccine in guinea pigs. rPA combined with the Ribi adjuvant was found to provide 100% protection against challenge.

Doxycycline or ciprofloxacin prophylaxis and therapy against experimental Yersinia pestis infection in mice.

The efficacy of doxycycline and ciprofloxacin against an experimental plague infection was assessed by comparing the median lethal dose (MLD) of Yersinia pestis in antibiotic-treated and untreated mice. The MLD of Y. pestis GB strain in untreated mice by the intra-peritoneal route was 23 cfu. If ciprofloxacin dosage (20 or 40 mg/kg twice daily) was initiated 48 h before infection, it afforded complete protection against an intra-peritoneal challenge of 5.24 x 10(7) cfu. Ciprofloxacin therapy initiated 24 h post-challenge was less protective, the MLD was raised to 2.0 x 10(5) and 2.2 x 10(5) cfu for 40 and 20 mg/kg respectively. Doxycycline dosage (40 mg/kg twice daily) initiated 48 h prior to infection raised the MLD to 1.6 x 10(4) cfu, but other prophylactic and therapeutic regimes were ineffective against challenges greater than 6.76 x 10(2) cfu. Ciprofloxacin may therefore be a useful antibiotic to consider for the treatment of plague.

A comparison of Plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a murine model.

The median lethal dose (MLD) of a pathogenic strain of Yersinia pestis was established by three routes of administration in three strains of mouse. There was no significant difference between the MLDs in the different strains of mouse. The MLD by the subcutaneous route in Balb/C and an outbred line was approximately 1 c.f.u.; the MLD following intraperitoneal administration was tenfold higher. There were significant differences in the mean times to death after administration of the challenge by different routes. The relative efficacy of a live attenuated vaccine strain of Y. pestis (EV76) was compared with that of the formaldehyde-killed vaccine (Plague vaccine, USP). EV76 protected against high challenge doses (up to 5.75 x 10(6) MLD), though immunized animals showed side effects of varying severity. The killed vaccine was less effective in terms of dose-protection (deaths occurred after challenge with 4000 MLD) and several of the vaccinated animals suffered sub-lethal, plague-related sequelae to the challenge.

Evaluation of the Biolog system for the identification of Bacillus anthracis.

The potential of the Biolog system for the identification of Bacillus anthracis was evaluated. In-house generated databases allowed the correct identification of 19 of 20 isolates of B. anthracis within 24 h. Five strains of the closely related B. cereus/thuringiensis group were misidentified as B. anthracis. For this reason the test could only serve as a primary screen with further testing being required to confirm identity. In addition 20% of all the strains of bacilli examined during the study gave unreadable reaction profiles due to false-positive reactions.

Formaldehyde Solution Effectively Inactivates Spores of Bacillus anthracis on the Scottish Island of Gruinard.

Gruinard Island was heavily contaminated with the spores of virulent Bacillus anthracis during biological weapons trials in World War II. However, an extensive survey in 1979 showed that most of the island was not contaminated. In the early 1980s, a more intensive survey revealed that the contamination was largely confined to the top 8 cm of the soil in a 2.6-ha area of the 211-ha island. Small-scale tests showed that the spores could be inactivated by drenching the soil with fluid biocides. A solution of 5% formaldehyde in seawater applied by surface spray to each square meter of ground was shown to be the most effective treatment and was utilized for large-scale decontamination of the affected areas. Following this treatment, extensive sampling revealed that most of the spores of B. anthracis had been inactivated. Isolated pockets of surviving spores were treated further. A flock of sheep was then allowed to graze over the entire island for 5 months; none contracted anthrax.

Evaluation of Bacillus subtilis strain IS53 for the production of Bacillus anthracis protective antigen.

An asporogenous strain of Bacillus subtilis, IS53, transformed with plasmid pPA102, produces the protective antigen (PA) component of the tripartite toxin of B. anthracis. Addition of yeast extract was required to support growth and PA production in all the media examined. Protective antigen expression was down-regulated during exponential growth and extracellular proteases caused marked degradation of the mature protein.

The development and assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis.

Two DNA probes and a number of oligonucleotide probes were designed from the virulence factor genes of Bacillus anthracis. These probes were tested for specificity against 52 B. anthracis strains and 233 Bacillus strains encompassing 23 other species. A rapid slot blotting technique was used for screening the large numbers of isolates involved. All probes tested appeared to be specific for B. anthracis under high stringency conditions. These probes could differentiate between virulent and avirulent strains. The probes were also applied to the detection of B. anthracis in routine environmental and clinical samples. A non-radioactive hybridization and detection system based on digoxigenin-11-dUTP was developed.

Use of a zwitterionic detergent for the restoration of the antibody binding capacity of immunoblotted Francisella tularensis lipopolysaccharide.

A method for the partial restoration of the antibody binding capacity of Francisella tularensis lipopolysaccharide (LPS) following denaturation (dissociation) in boiling sodium dodecyl sulfate (SDS) is described. The method relies on the presence of a zwitterionic detergent in the matrix of an SDS-polyacrylamide gel and in the transfer buffer during an immunoblot. F. tularensis LPS, which had lost its earlier capacity to bind to a particular monoclonal antibody in the normal blot procedure, did bind following the addition of the zwitterionic detergent to the polyacrylamide gel and transfer buffer. A number of detergents were tested but most success in restoring antibody binding was achieved with Zwittergent 3-08. This simple modification to the immunoblot procedure proved helpful in identifying a monoclonal antibody specific to hot phenol-extracted F. tularensis LPS.

Production and characterization of monoclonal antibodies directed against the lipopolysaccharide of Francisella tularensis.

Two monoclonal antibodies (FT14 and FT2F11) directed against the lipopolysaccharide (LPS) of Francisella tularensis were produced for use in tests to detect the organism in environmental samples and clinical specimens. The specificity of the antibodies was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Both antibodies detected LPS from F. tularensis by ELISA, but only one antibody, FT14, was serologically active in an immunoblot. Treatment of the LPS with detergents prior to ELISA eliminated its binding to FT2F11 but not FT14. Qualitatively, both antibodies detected 10 different strains of F. tularensis by ELISA, but quantitatively, FT14 gave a detectable reaction with 10(3) organisms, whereas FT2F11 was able to detect only 10(5) organisms. FT14 did not cross-react with LPS from a range of other gram-negative species of bacteria, whereas FT2F11 cross-reacted against Vibrio cholerae LPS. Neither antibody showed cross-reactions when entire gram-negative organisms were used as antigens. In a competition ELISA, the two monoclonal antibodies were shown to compete for different epitopes. FT14 was strongly inhibited by purified O side chain from F. tularensis LPS, but FT2F11 was only weakly inhibited. It was inferred from those results that FT14 is directed against the O side chain and that FT2F11 is directed against the core.

Factors affecting the germination of spores of Bacillus anthracis.

Spores of Bacillus anthracis germinated poorly at high cell densities unless the alanine racemase inhibitor O-carbamyl-D-serine was added to the germination medium. Spores derived from a variety of strains of B. anthracis germinated optimally at 22 degrees C. No correlation was found between rate of spore germination and virulence or between susceptibility of animal species to anthrax and spore germination rate using sera from those animals as the germination medium.

Development of antibodies to protective antigen and lethal factor components of anthrax toxin in humans and guinea pigs and their relevance to protective immunity.

A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies in serum to the protective antigen (PA) and lethal factor (LF) components of anthrax toxin. Current human vaccination schedules with an acellular vaccine induce predictable and lasting antibody titers to PA and, when present in the vaccine, to LF. Live spore vaccine administered to guinea pigs in a single dose conferred significantly better protection than the human vaccines (P less than 0.001), although they elicited significantly lower (P less than 0.0005) anti-PA and anti-LF titers at time of challenge with virulent Bacillus anthracis. Substantial anti-PA and anti-LF titers may not, therefore, indicate solid protective immunity against anthrax infection. The ELISA system was also shown to be capable of detecting anti-PA and anti-LF antibodies in the sera of individuals with histories of clinical anthrax. The advantage of ELISA over the Ouchterlony gel diffusion test and indirect microhemagglutination assay are demonstrated. There was a highly significant degree of correlation between ELISA and the indirect microhemagglutination assay (P less than 0.0005); but ELISA was markedly superior in terms of reproducibility, reliability, specificity, and simplicity in performance and stability of the bound antigen.

Decontamination of Bacillus anthracis on Gruinard Island?

In experiments on Gruinard Island 40 years ago small bombs containing spores of Bacillus anthracis were suspended from a gantry and detonated, producing widespread contamination of the island's surface. Recently, analysis of soil samples has shown that the area where the spores can now be detected is small enough to be considered for decontamination. We investigated the effect of treating the soil with sporicidal chemicals, namely, potassium permanganate, formaldehyde, glutaraldehyde, Cidex ('activated' glutaraldehyde, Surgikos), dodecylamine and peracetic acid.

Mycoplasmal adherence with particular reference to the pathogenicity of Mycoplasma pulmonis.

Various eucaryotic cells adhere to colonies of some mycoplasmas (adsorption). The chemical nature of the receptors on the cells is not the same for all mycoplasma species, nor are the binding sites on different mycoplasmas the same. Some receptors comprise sialic acid, but in the case of Mycoplasma pulmonis, for example, attachment to cells is not mediated in this way. Nevertheless, adherence seems to be an important factor in the pathogenicity of this mycoplasma. Strain JB caused pneumonia in mice when inoculated intranasally, and colonies of this strain on agar absorbed erythrocytes (hemadsorption) strongly. After multiple passes in mycoplasma liquid medium, the strain lost its hemadsorbing capacity and also its mouse virulence, suggesting that the ability to attach to cells is virulence factor. Examination by electron microscopy of the virulent mycoplasma and its induced avirulent form after ruthenium-red staining showed that the stain was less thick on the surface of the avirulent form. In addition, the protein pattern of the avirulent mycoplasma, demonstrated by polyacrylamide gel electrophoresis, was deficient in three bands. These observations suggest that a glycosylated protein may form the binding site on M. pulmonis organisms that mediates their attachment to cells.

Enhanced growth of T-strain mycoplasmas with N-2-hydroxyethylpiperazone-N'-2-ethanesulfonic acid buffer.

The small size of T-strain mycoplasma colonies obtained on agar medium has been a drawback to the study of these organisms. The incorporation of 0.05 mN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), a new hydrogen ion buffer, into agar medium at pH 6.8 led to the production of colonies at least 60% greater in diameter than those obtained on media without HEPES at pH 6.5 or 6.0. In addition, the colonies often had the "fried-egg" appearance typical of "classical" large-colony-forming mycoplasmas. The addition of HEPES to liquid medium in the presence of 0.1% urea resulted in a slightly higher number of viable organisms and a corresponding increase in ammonia production. Rapid death of the mycoplasmas occurred on continued incubation of the liquid medium cultures even in the presence of HEPES. The larger colony size facilitated the study of hemadsorption and tissue-culture cell adsorption. Preliminary results of such tests in which these phenomena have been demonstrated are presented. The advantages and disadvantages of having larger colonies are discussed, and the terminology of T-strain mycoplasmas is considered in the light of the present findings.