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Title: Characterization of Dermatophilus congolensis grown in vitro
Author: El-Jack, M. A.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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The aims of this study were i) To optimize a liquid culture media for D. congolensis, ii) to develop methods of preparing and examining excretory-secretory products (ESP) in culture filtrate, iii) To ascertain the importance of antigens in ESP, released by D. congolensis hyphae in vitro, in immune response to D. congolensis infection. Following initial trials, the basic medium used was a commercial preparation of RPMI 1640 containing Hepes buffer. Several chemical supplements were examined, alone and in combinations for their effect on the growth of two D. congolensis isolates: A Scottish isolate (A5N) from a case of lumpy wool in a sheep and a Ghanian isolate (Gh'89) from a case of bovine dermatophilosis. An optimal synthetic serum free liquid culture medium was obtained by supplementing RPMI 1640 with 0.001 per cent ferrous sulphate, 0.05 per cent sodium pyruvate and 0.025 per cent sodium metabilsulfite (RPMI-FPM). In this medium the growth of the two isolates was similar except that the Scottish isolate rarely completed its life-cycle. Excretory-secretory products (ESP) of the two isolates were prepared from supernatants of cultures in serum-free liquid medium. The peptides in ESP were separated by SDS-PAGE and their relative molecular weights (MWT) calculated. The quantity of protein was greater in 48 hour than 24 hour cultures, the former was used to characterize the ESP. The ESP of A5N contained 15 peptides with MWT from 15-160 kDa. The ESP of Gh'89 contained 15 peptides of approximately (± 2 kDa) the same MWT as those in A5N ESP, a further five peptides in the range 100-200 kDa and two at 94 and 82 kDa. The molecular weight peptides (>90 kDa) of both isolates were susceptible to the action of proteases in the ESP, they were present only in samples containing protease inhibitors. In terms of quantity the dominant peptides in both isolates had MWT 67/70 and 54/56 kDa, the latter was not present in samples prepared by ultrafiltration. Both isolates consistently produced a group of five bands having MWT between 24 and 33 kDa. Doublets of peptides were more common in Gh'89 ESP than A5N ESP, occurring at for example 25/26 kDa and 30/31 kDa. Comparison of ESP peptide profiles with those from sonicated hyphae and filaments showed that the ESP are not products of cell lysis or breakdown of cell walls.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available