Evolutionary relationship between Trypanosoma evansi and trypanosoma brucei with respect to specific mitochodrial antigen and phenotype knockout analysis
Obanda, Benear Apollo
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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 Genbank® 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.