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Title: Haemagglutination and adhesion of Bordetella pertussis
Author: Burns, Kathleen A.
ISNI:       0000 0001 3510 9717
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1981
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The object of this work was to define the role of surface structures of Bordetella pertussis, particularly the fimbrial haemag-glutinin (F-HA), in the virulence of the bacteria and in their ability to adhere to the epithelium of mouse lung. Preparatory to this, it was first necessary to investigate the production of F-HA by different strains and under different cultural conditions. In agreement with recently published work, it was shown that B. pertussis produces two haemagglutinins (HA), one of which is associated with the toxic factor pertussigen and the other with the fimbriae. The F-HA is distinguished from pertussigen in that haemagglutination (HA) caused by it is inhibited by cholesterol. Cells grown in shaken liquid culture showed little evidence of F-HA as determined by HA tests and electron microscopy of the bacteria. In static cultures, both the cell-associated and cell-free HA were cholesterol-sensitive,and were associated with fimbriae visible in the electron microscope. Highly purified F-HA was obtained by passing the supernate of a 5 day static culture through CM-Sepharose CL6B, concentrating the eluted protein and adsorbing the F-HA specifically onto cholesterol- containing liposomes. This liposome adsorbed protein (LAP) was used as a vaccine in mouse protection tests, and to raise specific antiserum. The fraction left after adsorbing the F-HA onto the liposomes had a high histamine sensitising activity (HSF). The purified F-HA (LAP) when given to mice intraperitoneally (i.p.) two weeks before intracerebral (i.e.) challenge with approximately 200 LD50, of live B. pertussis strain 18323 showed protective properties. The fraction with high HSF activity also protected the mice. This HSF or pertussigen fraction was toxic at around 6mug/mouse. Antiserum raised against the LAP (F-HA) was also found to protect mice passively against i.e. and intranasal (i.n.) challenge. An adhesion assay for measuring the ability of B. pertussis to adhere to (or be retained in) the airways within mouse lungs was developed. Adhesion was found to be rapid over the first few minutes of. contact, followed by a slower rate and with a maximum level of adhesion after 30 min. C mode, phase IV and large-colony variants of B. pertussis, which were of low virulence, showed a low level of adhesion to the lungs. There was also difference between strain 77/18319 which is i.n.-virulent but not i.c.-virulent, and strain 18323 which is i.c.-virulent but less virulent i.n., in each case there was a parallelism between the adhesion levels and lung virulence. This may indicate that F-HA and adhesion are more important in lung infections than in brain infections of mice. Although HA by fimbriae was inhibited by cholesterol, adhesion and virulence were not inhibited, as judged by failure of cholesterol in the bacterial suspension to influence the results of these tests. Also cells from 48 h shake cultures were virulent, yet were HA-negative. Therefore there does not appear to be a simple direct relationship between the presence of fimbriae, as indicated by HA, and adhesion and virulence. In conclusion, it seems that fimbriae are involved in adhesion, perhaps by rendering the cells more hydrophobic, although the evidence is not fully conclusive. When used as an antigen, fimbriae protected mice against both i.n. and i.e. infections, and may therefore protect man. Pertussigen was found to be a very potent protective factor against i.e. challenge, and a reliable method for detoxifying it should be developed. It is suggested that a possible improved pertussis vaccine for human use might consist of purified F-HA mixed with detoxified pertussigen.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Microbiology