Leishmania mexicana and Leishmania major: Attenuation of wild type parasites and vaccination with attenuated lines

Daneshvar, Hamid (2001) Leishmania mexicana and Leishmania major: Attenuation of wild type parasites and vaccination with attenuated lines. PhD thesis, University of Glasgow.

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Abstract

The attenuated cell-lines of Leishmania mexicana Wild Type (L. mexicana WT) and Leishmania major Wild Type (L. major WT) known as L. mexicana Hamid's line (H- line) and L. major H-line, respectively have been established under the pressure of gentamicin which was routinely added to the medium to prevent bacterial contamination of promastigote culture. The mechanism by which gentamicin, an aminoglycoside, attenuates L. mexicana WT is unknown. Following culture of promastigotes of L. mexicana WT (20 passages) and L. major WT (11 passages) in HOMEM medium supplemented with 10% (v/v) FCS and gentamicin at 20 pg/ml, promastigotes of the two strains formed attenuated lines. L. mexicana H-line was developed on four separate occasions with the same procedure and was stable in gentamicin-free medium for 23 weeks. There was no significant difference between the growth rate of promastigotes of L. mexicana H-line and L. mexicana WT in vitro. 12% of stationary phase promastigotes of L. mexicana H-line were longer than that of promastigotes of L. mexicana WT. Total lysate protein of stationary or log phase promastigotes of L. mexicana H-line and WT, on 10-20% SDS-PAGE gradient gel, showed some differences between protein expression of the attenuated cell line and L. mexicana WT. Two bands were detected around 66 kDa with stationary or log phase promastigotes of L mexicana WT, whereas one (possibly two) line is absent with stationary and log phase promastigotes of L mexicana H-line. The optical density of proteins in lysates of stationary phase promastigotes of two lines of L. mexicana separated using SDS-PAGE was displayed with a Lane profile graph using Lab Works Image Acquisition and Analysis Software (UVP Laboratory products). The optical density of protein of lysate of L. mexicana H-line showed just one peak of protein with high concentration, whereas three peaks of protein are found in the same position of graph of Z. mexicana WT. The comparative proteome analysis of the two lines of L. mexicana using high- resolution techniques has been done using 2-dimentional electrophoresis (2-DE). Both lines of Leishmania comprise patterns with a high density of spots in the gel with pH range pH 4-7. The results of proteome analysis of promastigotes of the two lines of L. mexicana suggest that adaptation of L. mexicana H-line to grow in the presence of gentamicin has involved change in protein expression. The proteome analysis of XIX patterns of two lines of L. mexicana reveals high similarity and significant differences between attenuated line of L. mexicana and L. mexicana WT pattern have been found. One spot of pattern of L. mexicana WT was shifted to less acidic position in the pattern of L. mexicana H-line and one spot was absent from the pattern of L. mexicana H-line. Two spots were foimd in the L. mexicana WT gel, whereas the expression of these proteins by promastigotes of L. mexicana H-line decreased. The ability of promastigotes of L. mexicana H-line to infect BMMs was similar to that of promastigotes of L. mexicana WT. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Stephen Phillips
Keywords: Parasitology
Date of Award: 2001
Depositing User: Enlighten Team
Unique ID: glathesis:2001-72384
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2019 15:12
Last Modified: 24 May 2019 15:12
URI: http://theses.gla.ac.uk/id/eprint/72384

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