Development of PCR based techniques for the characterisation of Trypanosoma brucei strains from East Africa

Tilley, A

Abstract

Human trypanosomiasis in east Africa, caused by Trypanosoma brucei rhodesiense,
occurs in specific foci that alternate between endemic and epidemic states.
Identification of T.b. rhodesiense is complicated as it also circulates in cattle
reservoirs, alongside the morphologically identical, non-human-infective,
Trypanosoma brucei brucei. The spread of disease away from one traditional focus in
Southeast Uganda has been linked to cattle restocking from infected areas. The
importance of identifying, typing and tracking human-infective trypanosomes in cattle
is therefore evident and rapid sensitive approaches and techniques are required. In
this work, two molecular techniques were developed for the characterisation of
trypanosome strains and used to address epidemiological questions pertaining to
sleeping sickness.
Analysis of restriction endonuclease polymorphism variation in PCR generated
intergenic regions of the ribosomal gene locus (PCR-RFLP) between trypanosome
stocks showed very low levels of discrimination. Although this analysis separated
stocks into groups reflecting location and human-infectivity, the discriminatory ability
was based solely on variation using a single restriction endonuclease.
The second, more successful, technique measured length and positional variation of
the trypanosomal mobile genetic element RIME. Variation in length classes of RIME
between trypanosome stocks was not observed. However, development of a novel
single primer technique for the analysis of positional variation of RIME (termed
MGE-PCR) revealed considerable variation between stocks. A key novelty of MGE-
PCR is that positional variation could be analysed without sequence knowledge from
xinRIME flanking regions. Phylogenetic analysis of this variation revealed a division of
stocks into human-infective and non-human-infective groups, which were further sub-
divided to show variation between strains. This technique demonstrated that human
infective isolates taken from the Uganda sleeping sickness focus are distinct from
those in the Zambian focus. Furthermore, it has demonstrated the possibility the
human infective trypanosomes can be found in Kiboko, Kenya, a region free of
sleeping sickness.

Citation

Tilley, A. Development of PCR based techniques for the characterisation of Trypanosoma brucei strains from East Africa. (Thesis). Salford : University of Salford

• Thesis Type: Thesis
• Deposit Date: Oct 3, 2012
• Award Date: Jan 1, 2002