Antigens of Leptospires

The antigens of Leptospira (biflexa, Waz) were investigated. The work was divided into four sections: Section 1. The whole leptospire. Section 2. The trypsin extractable antigens of the leptospire. Section 3. Separation of precipitating antigens of the leptospiral trypsin extract. Section 4. Monospecific antisera to individual precipitating antigens of the leptospire. Section 1. The whole leptospire Normal and trypsinized leptospires each gave rise to approximately the same level of agglutinating antibodies in rabbits. The early agglutinins were all 2-Me sensitive but in hyperimmune sera only 50% of agglutinating activity was destroyed with 2-Me. The 2-Me sensitive agglutinin was shown to be IgM while the 2-Me insensitive agglutinin was associated with a ¥ migrating 7S immunoglobulin, probably IgG. By immunizing with whole leptospires, precipitating antibodies against the axial filament were produced concurrently with agglutinating antibodies. Rabbit antileptospiral agglutinating sera always contained axial filament precipitins without which the serum did not agglutinate whole leptospires. This suggests that the axial filament is an agglutinating antigen of the whole leptospire. The early bleed axial filament precipitins were 2-Me sensitive but became 2-Me insensitive after day 6 when they were found in both 19S and 7S gel filtration fractions. Precipitating antibodies against other leptospiral trypsin extract (LTE) antigens did not appear until many days later and then usually not without further immunization. Their time of appearance varied between individual rabbits, as did the specificity of the various precipitating antibodies produced by different rabbits. The leptospiral microscopic agglutination test (MAT) was investigated for factors other than the serum antibody level which affected the agglutination titre of the serum. Both the concentration of leptospires and the percentage of viable cells in the antigen suspension affected the endpoint of the titration. However the age of the leptospiral suspension and the incubation time for the reaction mixture did not affect the endpoint of the titration. Section 2. The trypsin extractable antigens of the leptospire. Virtually all the agglutinating activity of rabbit antileptospiral serum could be removed by absorption with LTE. The LTE as an immunogen stimulated the same level of agglutinating antibodies in rabbits as did whole leptospires. Precipitating antibodies (including those against axial filaments and other antigens) were also produced. One particular rabbit antileptospiral serum (R198-72) produced 3 strong precipitin bands in immunodiffusion against LTE; 'axial filament', 'd' and 'e'. The LTE represented about 20% of the dry weight of the whole leptospire. About 1/5th of the LTE, including the precipitating antigens, was not dialysable. The LTE was mainly protein (73-101%) but contained considerable amounts of carbohydrate (10-12%). Extracts from different batches of cells showed substantial differences in chemical composition. No enzyme activity was detected in the LTE. Young leptospiral cultures (2 - 6 days) were marginally more susceptible to trypsin extraction (26 - 30% of dry weight extracted) than were older cultures (8 - 14 days) (19 - 22% of dry weight extracted). Furthermore, antigens 'd' and 'e' could be detected only in stationary phase leptospires and not in logarithmically growing cells. These antigens could not be detected in concentrated culture supernatants of either young or old cultures. Purified antigen 'd' was shown to be mainly protein and contained only 3% carbohydrate. Antigen 'd' was destroyed by papain, but not by trypsin, 'pronase' or lipase. Antigens 'e' and 'axial filament' could not be destroyed with any of these four enzymes. Antigens 'd' and 'e' were heat labile (100°C for 1 hour). Neither 'd' nor 'e' precipitin band stained specifically for lipid or polysaccharide. Trypsinized leptospires and the LTE were examined by by electron microscopy. Trypsin caused release of axial filaments and an uneven loss of outer cell wall material from the whole leptospire. Section 3. Separation of Precipitating antigens of the leptospiral trypsin extract. Gel-filtration, ion-exchange chromatography, electrophoresis, !so-electric point precipitation, ammonium sulphate fractionation and immunoadsorption were used in attempts to separate mixtures of precipitating antigens. Antigen 'd' was prepared in a purified form, free from other antigens, but antigen 'e' was not. Antigen 'd' was shown by gel-filtration to have a molecular weight of about 500,000. Section 4. Monospecific antisera to individual antigens of the leptospire. Monospecific antisera to individual precipitating antigens were usually prepared by immunizing rabbits with the washed immune precipitate in agar. Other methods involved immunizing with gel-filtration-column fractions and with antigens eluted from immunoadsorbents. Rabbit antibodies produced against antigens 'd' and 'e' were initially 19S but later production changed to 7S, without any 19S component. This suggested that the antigens were protein rather than polysaccharide. Monospecific antisera against antigens 'd' and 'e' would not agglutinate whole leptospires, nor would they fix a detectable amount of complement when reacted with normal, trypsinized or sonicated leptospires. Anti-'d' and anti-'e' antibodies could be absorbed from serum with.whole leptospires from stationary phase cultures. When anti-'d' and anti-'e' sera were labeled with fluorescein isothiocyanate they specifically stained alcohol fixed leptospires on glass slides, but not unfixed leptospires in test tubes. Fluorescein labeled anti' whole leptospire' serum stained under both conditions. Leptospires pretreated with rabbit anti-'d' serum or rabbit anti-'e' serum could not be cross-linked (agglutinated) with goat anti-rabbit serum. However, leptospires treated with rabbit anti-'whole leptospire' serum could be further agglutinated with goat anti-rabbit serum. This was interpreted as indicating that antigens 'd' and 'e' were sub-surface protein antigens not involved in whole cell interactions. Further support for the hypothesis of a sub-surface location of antigens 'd' and 'e' was given by the inability of fluorescent anti-'d' or anti-'e' antisera to react with whole leptospires (unfixed) and their inability to agglutinate whole leptaspires. Antigens 'd' and 'e' of Leptospira (biflexa Waz) were detected by immunodiffusion in other leptospiral biflexa (saprophytic) serotypes, but not in pathogenic serotypes. A survey of other leptospiral serotypes with fluorescent anti-'d' serum showed extensive crossreaction. Reasons for this are discussed.