Browsing by Author "Ouma, J. O."
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Item Evaluation of Indirect Enzyme-Linked Immunosorbent Assay (Elisa) Systems For The(2007) Ouma, J. O.; Mwangi, J. M.; Mdachi, E. R.; Murilla, A. G.; Kenya Trypanosomiasis Research InstituteAfrican animal trypanosomosis caused by Trypanosoma brucei Plimmer & Bradford 1899, T: congolense Broaden 1904 and T. vivax Ziemann 1905 remains one of the major constraints to health and productivity of cattle and other domestic animals in the tsetse infested areas of Tropical Africa. Owing to its varied clinical. Manifestations, diagnosis of trypanosomosis cannot be based on clinical signs alone (Nantulya 1990). Reliable diagnostic methods are a prelude to understanding the epidemiology of the disease and an important consideration when assessing the success of trypanosomosis control programmes. Conventional parasitological methods have a limited sensitivity and may lead to under-estimation of the prevalence of the disease (Paris et a/. 1982) More sensitive' diagnostic tests, including those for the detection of Trypanosoma specific antibodies (Luckins 1977) and antigens (Rae and Luckins, 1984; f'.iantulya et a/. 1992) have therefore been developed. Recently, four indirect ELiSAs have been developed, and their robustness and diagnostic performance evaluated (Rebeski et a/. 2000 a, b). One of the problems that has characterised users of the IAEA ELISA kits for bovine trypanosomosis has been that of maintaining the stability of the antigen during shipment to counterpart laboratories. This study utilised antigen pre-coated plates as a way to try and circumvent the problem. This paper reports on the validation of four indirect ELISA systems using antigen pre-coated polystyrene microplates. The usefulness of the current version of indirect ELISA as an epidemiological tool for studies in bovine trypanosomosis is discussed.Item Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B(2010) Ouma, J. O.; Njiru, Z. K.; Enyaru, C.J.; Kenya Trypanosomiasis Research Institute; Division of Health Sciences, School of Nursing and Midwifery, Murdoch University, Education Drive, Mandurah, WA 6210, Australia, Trypanosomiasis Research Centre (TRC), Kenya Agricultural Research Institute, Department of Biochemistry, Faculty of Science, Makerere University, Kampala, Uganda, Division of Health Sciences, School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch Drive, Perth, WA 6150, Austral, College of Veterinary Medicine, Central Mindanao University, University Town, Musuan, Bukidnon 8710, PhilippinesCamel Trypanosomiasis (Surra) is mainly caused by Trypanosoma evansi strains that express variable surface glycoprotein (VSG) RoTat 1.2. However, in Kenya a second causative strain that does not express RoTat 1.2 VSG (Trypanosoma evansi type B) has been identified, the prevalence of T. evansi type B largely remains unknown due to inadequate diagnostic assay. This work reports the development of a sensitive and specific diagnostic assay capable of detecting T. evansi type B based on the strategy of Loop-mediated Isothermal Amplification (LAMP) of DNA. The test is rapid and amplification is achieved within 20-25 minutes at 63 DC using a real time PCR machine. Restriction enzyme Alui digestion of the amplicon gave the predicted 83 bp and 89 bp sized bands and the LAMP product melt curves showed consistent melting temperature (T m) of 89Degree C. The assay analytical sensitivity is ~O.l trypanosomes/ml while that of classical. Per test targeting the same gene is 1 0 trypanosomes/ml. There was a 100% agreement in detection of the LAMP amplification product in real-time, gel electrophoresis, on addition of SYBR Green I, and when using chromatographic Lateral Flow Dipstick (LFD) format. The use of the LAMP test revealed nine more T. evansi type B DNA samples that were not initially detected through PCR. The robustness and higher sensitivity of the T. evansi type B LAMP assay coupled with the visual detection of the amplification product indicate that the technique has strong potential as a point-of-use test in surra endemic areas.Item Macrogeographic population structure of the tsetse fly, Glossina pallidipes (Diptera: Glossinidae)(2005) Ouma, J. O.; marquez, J. G.; Krafsur, E. S.; Kenya Trypanosomiasis Research InstituteTsetse flies are confined to sub-Saharan Africa where they occupy discontinuous habitats. In anticipation of area-wide control programmes, estimates of gene flow among tsetse populations are necessary. Genetic diversities were partitioned at eight microsatellite loci and five mitochondrial loci in 21 Glossina pallidipes Austin populations. At microsatellite loci, Nei's unbiased gene diversity averaged over loci was 0.659 and the total number of alleles was 214, only four of which were shared among all populations. The mean number of alleles per locus was 26.8. Random mating was observed within but not among populations (fixation index FST = 0.18) and 81% of the genetic variance was within populations. Thirty-nine mitochondrial variants were detected. Mitochondrial diversities in populations varied from 0 to 0.85 and averaged 0.42, and FST = 0.51. High levels of genetic differentiation were characteristic, extending even to subpopulations separated by tens and hundreds of kilometres, and indicating low rates of gene flow.Item Microgeographical breeding structure of the tsetse fly, Glossina pallidipes in south-western Kenya(2006) Ouma, J. O.; marquez, J. G.; Krafsur, E. S.; Kenya Trypanosomiasis Research InstituteThe origins of extant Glossina pallidipes Austen (Diptera: Glossinidae) populations in the ecologically well-studied Lambwe and Nguruman valleys in Kenya are controversial because populations have recovered after seemingly effective attempts to achieve high levels of control. The micro geographical breeding structure of the tsetse fly, G. pallidipes, was investigated by analysing spatial and temporal variation at eight microsatellite loci to test hypotheses about endemism and immigration. Samples were obtained at seasonal intervals from trap sites separated by 200 m to 14 km and arranged into blocks. G. pallidipes populations nearest to Lambwe and Nguruman also were sampled. Spatial analysis indicated that genetic differentiation by genetic drift was much less among trapping sites within Lambwe and Nguruman (FST :::; 0.049) than between them (FST = 0.232). FST between Serengeti and Nguruman was 0.16 and FST between Kodera Forest and Lambwe was 0.15. The genetic variance in G. pallidipes explained by dry and wet seasons (0.33%) was about one-fifth the variance among collection dates (1.6%), thereby indicating reasonable temporal stability of genetic variation. Gene frequencies in Kodera and Serengeti differed greatly from Lambwe and Nguruman, thereby falsifying the hypothesis that Lambwe and Nguruman were repopulated by immigrants. Harmonic mean effective (= breeding) population sizes were 180 in Lambwe and 551 in Nguruman. The genetic data suggest that G. pallidipes in Lambwe and Nguruman have been endemic for long intervals.Item New Polymorphic Microsatellites in Glossina pallidipes (Diptera: Glossinidae) and Their Cross-Amplification in Other Tsetse Fly Taxa(2006) Ouma, J. O.; marquez, J. G.; Krafsur, E. S.; Kenya Trypanosomiasis Research InstituteTsetse flies (Diptera: Glossinidae) are discontinuously distributed throughout sub-Saharan Africa (Rogers and Robinson, 2004). They feed exclusively on blood and transmit hemoflagellate protozoan parasites. Trypanosomes that cause sleeping sickness in humans and nagana in livestock. Tsetse taxa are subdivided into three subgenera, Glossina (morsitans group), Nemorhina (palpalis group), and Austenina (fusca group). Glossina pallidipes belongs to the morsitans group and is among the most important vectors of trypanosomes.Item Patterns of genetic diversity and differentiation in the tsetse fly Glossina morsitans morsitans Westwood populations in East and southern Africa(2007) Ouma, J. O.; marquez, J. G.; Krafsur, E. S.; Kenya Trypanosomiasis Research InstituteGenetic diversity and differentiation within and among nine G. morsitans morsitans populations from East and southern Africa was assessed by examining variation at seven microsatellite loci and a mitochondrial locus, cytochrome oxidase (COl). Mean COl diversity within populations was 0.63 ± 0.33 and 0.81 taken over all populations. Diversities averaged over microsatellite loci were high (mean number of alleles/locus: 7.4; mean HE 65%) in all populations. Diversities averaged across populations were greater in East Africa (mean number of alleles = 22 ± 2.6; mean he = 0.773 ± 0.033) than in southern Africa (mean number of alleles = 18.7 ± 4.0; mean he = 0.713 ± 0.072). Differentiation among all populations was highly significant (RST = 0.25, FST = 0.132). Nei's Gil statistics were 0.09 and 0.19 within regions for microsatellites and mitochondria, respectively; between regions, Gil was 0.14 for microsatellites and 0.23 for mitochondria. GST among populations was 0.23 for microsatellite loci and 0.40 for mitochondria. The F, G and R statistics indicate highly restricted gene flow among G. m. morsitans populations separated over geographic scales of 12-917 km.
