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Title: Inactivation of Bordetella pertussis by rat lung lavage fluids (LLF)
Author: Al-Fellah, Giamal Nouri
ISNI:       0000 0001 3405 5112
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1997
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Soluble antibacterial substance(s) in lung lavage fluid (LLF) from healthy normal rats killed phase I Bordetella pertussis in vitro. Phase IV B. pertussis was insusceptible. LLF that was active towards phase I organisms was obtained from rats of the Sprague Dawley, Lewis, Brown Norway, and Hooded Lister strains. Two strains of B. pertussis were mainly used: B. pertussis 18-323 for viable count experiments and B. pertussis lux for tests with the luminometer. The former experiments were more time consuming but more reliable; luminometry, although rapid, was complicated by substances that affected the luciferase reaction. Electron microscopy showed that LLF caused severe damage to the B. pertussis cells, with loss of internal contents Of the several mammalian species tested, the rat yielded LLF of the highest bactericidal activity (BA) towards B. pertussis. The other species LLF were: human, mouse, horse, dog, chicken, rabbit, sheep and calf. Typically rat LLF could be diluted 24 times and still exhibit BA. For assessment of the BA of LLF, both positive and negative control fluids were used. Normal rat serum, which killed the bacteria rapidly, was used for the former, and phosphate buffered saline (PBS), cyclodextrin liquid (CL) medium and casamino acids (CAA) for the latter. The half-life of B. pertussis at 37°C in PBS was 149 min and in CAA 155 min. CL medium allowed growth. To obtain LLF, four different methods of euthanasia were explored; the highest BA was in LLF obtained by anaesthetising the rats with halothane/ 02, then heart puncture for blood, and finally cervical dislocation. The least active LLF was obtained after CO2 euthanasia. LLF from normal rats was separated by ultracentrifugation at 55,000 g into supemate and surfactant (pellet) fractions. About 95 % of the BA towards B. pertussis 18-323 and lux was located in the surfactant fraction. No BA towards 18-323 was found in commercially-produced, protein-free artificial surfactant used for alleviating respiratory distress syndrome in premature infants . Physical, chemical and biochemical treatments of LLF and its fractions suggested that the substance(s) responsible for the BA towards B. pertussis 18-323 and lux are probably long-chain fatty acids, protein and phospholipids. There appeared to be no involvement of antibody, complement and lysozyme in the BA of LLF towards B. pertussis. However, the lysozyme in LLF was able to kill and lyse Micrococcus luteus. LLF from rats convalescent after infection with phase I B. pertussis tended to have BA lower than that from normal animals. This suggested that one of the toxins of B. pertussis might inhibit the synthesis or release of the substance(s) responsible for BA from the secreting cells in the lungs. B. parapertussis was sensitive to the BA of both surfactant and supemate of LLF from normal rats, while B. bronchiseptica was resistant. Other LLF-resistant species were Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli. B. pertussis 18-323 was strongly sensitive to the BA of long-chain fatty acids, of which the most active was arachidonic (C20:4). The BA of LLF from normal rats could be replicated by a mixture of fatty acids from C14 to C20 at a concentration similar to that found in the LLF itself. The reduced BA in LLF from convalescent rats might be due to the action of pertussis toxin on type II pneumocytes which are principally involved in production and recycling of surfactant. Further studies on the possible effect of PT on this system might provide valuable insights into pathogenic mechanisms in pertussis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Whooping cough