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