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SUMMARYFactors concerned in demonstrating haemadsorption and haemagglutination and their occurrence among different mycoplasmas were investigated. Haemadsorption occurred best to colonies which had recently developed on agar at pH 6.5. Mycoplasmas isolated from various bird and animal sources, e.g. Mycoplasma gallisepticum, M. agalactiae, M. bovigenitalium and M . pulmonis, haemadsorbed with erythrocytes from a wide range of species. However, not all strains within a serotype haemadsorbed. Thus, the 'Negroni' strain of M . pulmonis did not. Haemadsorption could be inhibited by crowding of colonies on agar and by the addition of specific antiserum to the colonies. Generally, antiserum titres obtained by haemadsorption inhibition were low in comparison with those obtained by metabolic inhibition, and haemadsorption inhibition was not useful as a routine serological technique.The development of the haemagglutinin of Mycoplasma gallisepticum in liquid medium was studied in detail; a change in the pH value of the medium could be used as an index of its development and it was intimately associated with the organism. The centrifuged deposits of other mycoplasmas, from birds, cattle, goats, man, rodents and pigs, and which were grown in liquid medium also haemagglutinated, but generally to low titre. Haemagglutination occurred best in U-shaped cups at 37' and at pH 6.5-7-0 and could be inhibited by specific antiserum. There was lack of correlation between haemadsorption and haemagglutination; both these phenomena were exhibited by some mycoplasmas, others haemadsorbed only, and still others haemagglutinated only.

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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.

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 vaccines administered to guinea pigs in a single dose conferred significantly better protection than the human vaccines (P < 0.001), although they elicited significantly lower (P < 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 < 0.0005); but ELISA was markedly superior in terms of reproducibility, reliability, specificity, speed, and simplicity in performance and stability of the bound antigen.

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.

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.