Evolutionary relationship between Trypanosoma evansi and trypanosoma brucei with respect to specific mitochodrial antigen and phenotype knockout analysis
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Date
2005
Authors
Obanda, Benear Apollo
Journal Title
Journal ISSN
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Publisher
Kenyatta University
Abstract
Trypanosomiasis is a disease caused by parasitic protozoans of the genus Trypanosoma.
The agents of the disease are obligate extracellular parasites that occur in blood,
cerebrospinal fluid and tissue fluids. Trypanosoma brucei causes sleeping sickness and
agana in sub-Saharan Africa. Trypanosoma evansi causing surra is endemic in Asia,
Middle East northern Africa including Northern Eastern Kenya. Salivarian
trypanosomiasis is one of the most important and widespread diseases of domestic
animals and man in the world. The causes of the re-emergence of this disease include
widespread civil war, declining economies, reduced health financing and the dismantling
of disease control programs. The current drugs in use are toxic and not effective because
of drug resistance, hence, the need for developing new drugs. The study objective was to
establish the evolutionary relationship between T brucei and Tevansi, with respect to
cell differentiation life cycle specific antigens and phenotype knockout analysis. PCR
was used to compare genes encoding mitochodrial protein of Tevansi IL 1695, Tevansi
IL 1934 and Trypanosoma brucei rhodesiense IL2343. Plasmid construction, preparation
of plasmid DNA was done using alkaline lysis method. Extraction and purification of
plasmid was by QIAGENR plasmid protocol. Cell line of T evansi and T brucei for RNA
interference experiments were established. Electroporation was by Gene-Pulse machine
for generation of knockout phenotypes. Statistical analysis was by Student's t-test.
Tevansi IL1695, Tevansi III934 and T b. rhodesiense IL2343 contain all the five genes
for mitochodrial protein in their genomes. MP 48 and MP 52 RNA editing ligases genes
were identified in Trypanosoma evansi. Specific RNA ligases MP 48 630 bp and MP 52
560bp primers were developed. These primers specifically identify T evansi, T brucei
and T equiperdum from other organisms. Alignment of MP 48 and MP 52 gene
sequences obtained in T evansi and T brucei show 100 % homology. Comparisons of
MP 48 and MP 52 RNA ligase gene with data of closely related organisms available in
GenbankĀ® showed no significant homology with the RNA ligase sequences of
TcruziREL and L. majorREL2 sp. nor with the available sequences ofLt RNA ligase.
Multiple alignment of T evansi MP52 and MP 48 with related proteins show a perfectmatch
with T brucei and near-perfect match of genes with data of closely related
organisms available in Genbank. T evansi was able to use T7 promoter gene, to
recognize bacteriophage T7 RNA polymerase and produce RNA polymerase that
synthesize mRNA encoding Green fluorescent protein, that was observed as Green
fluorescent Tievansi. Approximately 40% of original populations of both the species were
killed due to RNA interference. There was no significant difference in the effect of RNA
interference in Tbrucei Gutat 3.1 and 'Levansi Tansui 13, using Student's t-test twotailed,
p> 0.05 at 95% confidence interval. These new sub genus specific primers can be
used as a diagnostic tool for monitoring pathogenic Trypanozoon parasites in humans,
domestic animal. The primers can also be used in epidemiological survey. The RNA
interference analysis identified MP48 and MP 52 RNA editing ligases as a drug target for
the development of novel therapeutics in treatment of sleeping sickness, nagana and
surra.
Description
Department of Microbiology, 127p. 2005