PHD-Department of Biochemistry and Biotechnology

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    Agrobacterium tumefacients mediated introgression of Nicotiniana Protein Kinase (NPK1) gene in selected Kenyan maize genotypes to enhance drought tolerance
    (2011-08-12) Muoma, John Vincent Omondi; Machuka, Jesse; Geoffrey Muluvi
    In the last decade, the production of maize has gone down because of biotic and abiotic constraints, and this has resulted to prevalent famine. Of the abiotic stresses, drought is the most important stress affecting productivity of maize in Africa leading to up to 70% crop loss and in certain cases total crop loss. Conventional breeding, molecular marker assisted breeding and genetic engineering have already had, and will continue to have, important roles in maize improvement. The rapidly expanding information from genomics and genetics combined with improved genetic engineering technologies offer a wide range of possibilities for enhanced maize production. Genetic engineering of plants has been achieved through direct uptake of naked DNA into target cells and via Agrobacterium mediated transformation. Agrobacterium mediated transformation is increasingly becoming the method of choice due to its ability to generate transformed events containing low copy insertions. However this mode of maize transformation is dependant on genotype, age and physiological condition of the target explant and the infecting strain of A. tumefaciens. For every successful transformation protocol a reproducible regeneration system and transformation by a reporter gene is a necessity. The optimal regeneration condition for the shoot tips and immature zygotic embryos was observed to be 9µM 2, 4-D, 8.88µM BAP supplemented with 296µM adenine and 9µM 2, 4-D respectively for calli maintenance and shoot induction. Root induction in case of shoot apices was alleviated by the use of 1.97µM indole-3- butyric acid while immature zygotic embryos readily formed roots on MS without hormone after a maturation step. Transient expression of GUS was used to assay the explants for transformation frequency considering embryogenic calli formation, shoot induction and root formation had been optimized in the regeneration step. In these experiments, 10 days old seedlings shoot apex derived calli exhibited GUS activity at a transformation frequency (TF) of 0-4.2% while the 15 days old immature zygotic embryos derived calli exhibited a higher TF of 613% GUS activity making immature zygotic embryos better explant for transformation of the selected genotypes. Immature zygotic embryos were thus preferably transformed for drought with a gene that codes for an upstream transcription signal factor MAPKKK cascade (NPK1) triggered under drought stress (DS). The transformation efficiency for the four genotypes was TL08 0.79%, DHO1 4.87%, DLC1 2.64% and PTL001 5.35%. The seeds of the transgenic events were harvested, planted and both DNA and RNA extracted from the T1 events for southern, northern, and RT-PCR analysis to check on copy numbers and expression levels of the NPK1 gene. The T1 plantlets of tropical inbred TL08-(2)4, single hybrid cross of a PTL001, a multiple cross hybrid DHO1 and a dry land cultivar DLC 1 genotypes were planted in the green house and assessed for morphological and physiological changes associated with increase in DS tolerance when under water stress condition. The results showed that NPK1 effectively enhanced drought tolerance in TL08-(2)4 and PTL001, and there was no significant morphological difference between transgenic controls (well watered) and transgenic tests (subjected to moderate drought stress) using Turkeys Kramer HSD (p<0.05). Overall, there was between 20-35% enhancements of yield on comparison of the transgenic stressed events with non-transgenic stressed control
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    Anti-Obesity, Cognitive Enhancing, Neurobehavioral, Antioxidant Effects And Phytochemical Profile Of Dichloromethane Leaf Extract Of Gnidia Glauca (Fresen)
    (Kenyatta University, 2019-11) Makori, Wycliffe Arika
    Obesity is a chronic metabolic disorder characterized by increased adipose tissue mass due to positive energy balance. The epidemic of obesity is currently on the rise probably due to increasingly sedentary lifestyles combined with easy availability of palatable, high-fat foods. It presents modifiable risk factor for cognitive impairements, anxiety and motor deficits. Globally, its prevalence has shown a startling increase in all age groups and have been associated with increased morbidity and mortality. Prescription of anti-obesity drugs can be useful adjuncts to diet and exercise for obese patients who have failed to achieve weight loss. However, these drugs are ineffective, not readily available, unaffordable and have been associated with adverse effects. Therefore, medicinal plants have drawn a sharp focus in recent times as complementary and alternative medicines owing to their biocompatibility, affordability and are assumed to be safe due to their long-term clinical application. Hereby, determination of therapeutic activities and identification of active principles from herbal prescriptions have become the prime focus in the validation of their folkloric usage and in drug discovery programs. The present study aimed to determine the anti-obesity effects, cognitive enhancing, neurobehavioral, antioxidant effects and phytochemical profile of dichloromethane leaf extract of Gnidia glauca. Obesity was experimentally induced by feeding the rats with prepared high-fat-diet (HFD) and water ad libitum for a period of 6 weeks. The in-vivo anti-obesity effects were determined by oral administration of the extract at dosage levels of 200, 250 and 300mg/kg body weight in HFD-induced obese rats from the 6th to 12th week along with HFD. Cognitive-enhancing effects of the extract in HFD-induced obese rats were determined using the Morris Water Maze experiment. The effects of the extract on neurobehaviors (locomotor activity, anxiety and exploration-like behaviors) in HFD-induced obese rats were tested using the Open Field Test. The in vitro antioxidant properties of the extract were determined using non-enzymatic assays. The phytochemical profile of the extract was determined using GC-MS. The results indicated that the extract exhibited potent anti-obesity effects in HFD-induced obese rats. It significantly reduced the body weight, organ weights, organo-somatic indices, anthropometric indices, the total fat content, adiposity index, atherogenic index as well as the lipid profiles (Triglycerides, Total Cholesterol, Low-Density Lipoproteins, and Very Low-Density Lipoproteins). However, it significantly increased levels of High-Density Lipoproteins. The extract increased levels of white blood cells, differential leukocyte counts, platelet count as well as red blood cells and related parameters. The extract improved hippocampal-dependent spatial learning and memory retention in HFD-induced obese rats. Moreover, it showed anxiolytic effects, increased spontaneous locomotor activity and exploration-like behaviors in HFD-induced obese rats. The extract also indicated in vitro antioxidant effects. The phytochemical analysis revealed the presence of 28 bioactive compounds in the extract. The anti-obesity effects, antioxidants activities, cognitive-enhancing effects and the improved locomotor and exploration-like behaviors could be attributed to the phytochemical compounds present in the plant extract. The present study, therefore, scientifically validated the traditional use of this plant and generated data that can serve as guide in the recruitment of the extract as a potential candidate for the synthesis of a new effective drug against obesity and associated complications. However, there is a need for bioassay-guided fractionation of bioactive compounds in Gnidia glauca. Besides, it is recommended to conduct comprehensive toxicity studies to establish the safety profiles of Gnidia glauca.
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    Antibacterial Efficacy and Safety of Selected Kenyan Medicinal Plants
    (2013-08-15) Abiba, Alfred Ogao
    Many disease-causing organisms have become resistant to conventional antibiotics. The toxicity and side effects of conventional drugs have also become increasingly unmanageable, while their costs are prohibitive. It has therefore become necessary to continue searching for alternative medicines to treat such diseases. The possibility of developing pharmaceutical industries using local raw materials should be looked into critically so that the cost of drugs can be affordable to the local people. This study involved screening of various medicinal plants commonly used by herbal medicine practitioners for bioactive compounds against bacteria. Standard bioassay methods were employed throughout this study. They included preliminary screening bioassay against human pathogenic bacteria strains using Agar Disc Diffusion method, Minimum Inhibitory Concentration (MICs), Minimum Bactericidal Concentration (MBCs), Time kill kinetics, phytochemical determination methods and one dose in vivo toxicity tests using mice. Hexane, dichloromethane, acetone and methanol extracts of ten (10) medicinal plants: Dichrostachyus. cinerea, Combretum molle, Euclea divinorum, Ficus sur, and other five plants were screened by preliminary bioassay against nine (9) human pathogenic bacteria strains (E. coli, ATCC 25922 S. aureus ATCC 85923, Pseudomonas aeruginosa NCTC 10662, Escherichia coli, Shigella flexinery, Pseudomonas aeruginosa, Staphlococcus aureus, Shigella dysenteriae and Klebsiella hospital isolates) using agar disc diffusion method. Acetone, methanol extracts of D. cinerea and C. molle showed remarkable inhibitory activity against all nine human pathogenic bacteria. Acetone and dichloromethane of E. divinorum and F. sur showed activity against all human pathogenic bacteria except P. aeruginosa and klebsiella. All extracts of six (6) plants were inactive against the test human pathogenic bacteria. MICs of acetone and methanol extracts of the four (4) plants D. cinerea, C. molle, and dichloromethane and acetone extracts of E. divinorum and F. sur were active against the test bacteria. MIC results showed that acetone and methanol extracts of D. cinerea, and C. molle gave very good low MICs ranges (0.3-2.5mg/ml), dichloromethane and acetone extracts of E. divinorum and F. sur gave MICs ranging between 7.5-100mg/ml. MBCs of acetone and methanol extracts of D. cinerea, and C. molle were as low as 0.3mg/ml while the highest being 5mg/ml. Phytochemical determination showed that both methanolic and acetone extracts of D. cinerea and C. molle contained alkaloids, tannins, steroids and triterpenoids, flavone glycones, anthrocyanins and reducing sugar. Kill kinetics studies of the bioactive organic extracts on the tested bacteria indicated that acetone extracts of D. cinerea, acetone extracts of C. molle, methanol extracts of C. molle, methanol extracts of D. cinerea at 4, 6 and 8mg/ml against Staphylococcus aureus ATCC 85923, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa NCTC 10662, displayed dose and time dependent rapid kinetics of the bacterial killing. Because the toxicity of a drug to the bystander host cells could render it unsuitable for therapeutic purposes, the toxicity of the active plant extracts against the tested bacteria was assessed. The reduced growth rate, organ weights, white and red blood cells counts, plus increased levels of biochemicals in the serum in plant extract treated animals relative to that of the controls, indicate that these extracts contained constituents with toxic effects on them. Such constituents include some of the phytochemicals present in these extracts. The mineral content may also cause toxicity but this was not the case with the measured minerals since their levels were below the recommended daily allowance except for potassium which was 67 to 141 times the recommended daily allowance.
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    Antidiabetic activities of ethyl acetate and aqueous extracts of pappea capensis, senna spectabilis, maytenus obscura, ocimum americanum and launaea cornuta
    (2014-08-18) Karau, Geoffrey Muriira
    Pappea capensis, Senna spectabilis, Maytenus obscura, Launaea cornuta and Ocimum americanum have been used traditionally in the management of several diseases including diabetes mellitus but their efficacy and safety after long term use is not scientifically evaluated. The aim of this study was to determine in vivo hypoglycemic activity and safety of aqueous and ethyl acetate extracts of leaves and stem barks from plants in male BALB/c mice. The extracts were orally screened for their hypoglycemic activity in alloxan induced diabetic mice using a glucometer. The safety of these extracts was studied in mice orally administered with 1g/kg body weight daily for 28 days by monitoring the changes in body weight, hematological and biochemical parameters. Mineral composition of the plant powders were estimated using energy dispersive X-ray fluorescence system (EDXRF); vitamins were estimated using HPLC system; while the types and quantities of phytochemicals present were assessed using standard procedures. Individual volatile phytochemicals in the extracts were determined using GC-MS system. Proximate composition of the dry plants parts powers was estimated using standard procedures while the energy content was determined using the Atwater factor. Oral administration: of both the leaf and stem bark ethyl acetate and aqueous extracts of P. capensis, S. spectabilis, M. obscura, L. cornuta and O. americanum in doses of 50, 100 and 200 mg/kg body weight lowered blood glucose; of the three tested doses of aqueous leaf and stem bark extracts of P. capensis and aqueous leaves extracts of M. obscura could not significantly alter the blood glucose levels in alloxan induced diabetic male BALB/c mice; of the aqueous extracts of L. cornuta at 50 and 200 mg/kg body weight dose raised the glucose levels beyond that of the diabetic control mice. Oral administration of aqueous leaves extracts of P. capensis, and O. americanum and ethyl acetate leaves extracts of S. spectabilis at 1 g/kg body weight daily in mice for 28 days significantly reduced the weekly body weight gain. The same oral dose of P. capensis (L and SB), S. spectabilis (L and SB), M. obscura (L and SB), L. cornuta and O. americanum altered the red blood cell count, hemoglobin levels, packed cell volume, mean cell hemoglobin concentration, mean cell volume, platelets; white blood cell count and their differential counts; altered the levels of blood urea nitrogen and the activities of alanine and aspartate aminotransferases, alkaline phosphatase and creatine kinase. The plants powders contained phenols, tannins, saponins, flavonoids, alkaloids, terpenoids, phylobatannins, cardiac glycosides, sterols, fatty acids and reducing sugars; vitamins C, E, B1, B3, retinol, β-carotene, lycopene and cryptoxanthin; minerals selenium, iron, zinc, copper, chromium, manganese, vanadium, molybdenum, cobalt and heavy metals nickel, aluminium, lead and mercury. The observed hypoglycemic activity and/or slight toxicity in the bioactive plants extracts could be associated with the phytonutrients and minerals present in these plants. This study recommends use of safe plants with antidiabetic activity as herbal remedies
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    Antidiabetic Properties and Safety of Aqueous Extracts of Triticum Aestivum, Hordeum Vulgare, Urtica Massaica and Cynanchum Viminale in a Rat Model
    (Kenyatta University, 2021) Kimani, Njoroge Gideon; Eliud MN Njagi; Nicholas . K. Gikonyo; Mathew P. Ngugi
    The primary goal in the management of diabetes mellitus is to realize normoglycemia as much as possible. In conventional medicine, oral insulin secretagogues and parenteral insulin are used as hypoglycemic agents. However, they have known side effects such as insidious hypoglycemia, lactic acidosis, liver injury and neurological deficit. On the other hand, numerous phytotherapeutics that have been used in traditional, complimentary or alternative medicine for the treatment of diabetes have not been evaluated for their effectiveness and or their toxicity effects. The objective of this study was to evaluate the in vivo glycemic and toxicity effects of aqueous extracts of Triticum aestivum, Hordeum vulgare, Urtica massaica and Cynanchum viminale. Diabetes was induced by intraperitoneally administering 10% alloxan monohydrate into Wistar rats. Rats with fasting blood glucose levels above 200 mg/L 72 hr post alloxan administration were used in this study to evaluate efficacy of doses of aqueous plants extracts against glibenclamide and insulin as reference drugs. Toxicity studies were done by oral or intraperitoneal administration of doses of aqueous plant extracts for 28 days. The rats were euthenized and blood taken for hematological and biochemical studies. Gross and relative organ weights were taken. Presence of minerals, phytonutrients and phytochemicals in the plants extracts was also established. A p < 0.05 was consindered statistically significant. The results obtained in this study indicates that both orally and intraperitoneally administered aqueous plants extracts used in this study exhibited varying degrees of blood glucose lowering effects. When orally administered, all the plants extracts did not have significant different effect on cardiac and pancreatic systems, liver and kidneys relative to the control. Significant variations in some gross organ and relative organ weights between the extracts administered and control rats though observed were not supported by haematological or biochemical changes hence not pathologically associated. However, statistically significant effects were recorded when some of the plants extracts were intraperitoneally administered compared to the controls. Flavonoids, alkaloids, saponins, steroids, cardiac glycosides, minerals and vitamins were found to be present in the aqueous plants extracts. In conclusion, the aqueous plants extracts evaluated in this study have demonstrated blood glucose reduction properties and validates their use in control and mitigation of diabetes mellitus. Further, results obtained on phytonutrients and bioactive secondary metaboloites assay indicate the presence of various functional groups. The synergistic activities of the various compounds present in the aqueous plants extracts in this study are attributable to the plant’s observed blood glucose lowering effects. When orally taken, none of the logarithmic doses of either of the plants extracts demonstrated any toxicity effects while some of the plants extracts exhibited toxicity when intraperitoneally administered. Thus, based on the findings of this study, continued use of the plants extracts and following proper monitoring and evaluation protocol in management of diabetes mellitus is recommended. However, caution should be observed in intraperitoneal administration of some of the extracts. Further isolation and structural characterization of the active constituents of phytochemicals of the plants and ellucidation of the actual mode of action is recommended. Translational clinical reseach is equally recommended on the background of the data obtained in this study to facilitate advances and the integration of the studied alternative botanical therapies for treating diabetes into modern medicine.
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    Banana Rhizosphere Microbial Diversity, Low-Cost Tissue Culture Bananas (Musa spp.) Micropropagation, Genetic Fidelity and Acclimatization Using Arbuscular Mycorrhizal Fungi
    (Kenyatta University, 2024-05) Wahome, Caroline Nyawira
    Banana production in Kenya predominantly involves smallholder farmers, yet current yields fall short of meeting local and international market demands due to constraints faced by these farmers among them being soil health and lack of clean planting materials. To address this issue, the study assessed banana cultivation practices in Kisii, Nyamira, and Embu counties, aiming to understand strengths and challenges within these regions. In this study, rhizospheric soil physicochemical properties and the diversity of microbial communities was assessed, shedding light on soil health status. Furthermore, the efficacy of a cost-effective micro-propagation technique for local banana cultivars was determined, considering contamination susceptibility and acclimatization efficacy of arbuscular mycorrhizal fungi on micropropagated banana plantlets. The study revealed a reliance on ineffective traditional propagation methods, with over 30% of the smallholder farmers facing a banana seedlings shortage. The soil physic-chemical parameters indicated signs of degradation, with low pH and nutrient deficiencies raising concerns with Embu County having the lowest pH measured (4.43). Soil metagenomic studies by NGS showed that proteobacteria and basidiomycota were the two most prevalent bacterial and fungus divisions, respectively. Based on the Shannon diversity indices, the highest bacterial and fungal community diversity was in soil samples obtained from Embu County and Kisii County respectively. To evaluate efficacy using low cost tissue culture technology, selected banana cultivars were utilized and results indicated the highest survival rate of 100 % in Kiganda, Muraru and Kienyeji banana cultivars established in BAP and IAA. Ng’ombe cultivar best responded to low cost micropropagation with the highest shoot establishment (80.5 %) and shoot emergence rates (75.5 %) producing the highest number of shoots per culture (10.61). To investigate clonal fidelity of the tissue culture plantlets Inter Simpler Sequence Repeats (ISSR) and the research demonstrated genetic homogeneity in banana seedlings generated via low cost tissue culture to the mother plant. During the micropropagation process the presence of bacterial and fungal contaminants in the tissue culture laboratory was noted with the most predominant bacterial and fungal contaminants being Microccoccus luteus and Cladosporium sp respectively. Additionally, broad-spectrum antimicrobial agents nalidixic acid (30 µg) and carbendazim+ triadimefom (0.3 µg and 0.6 µg) exhibited 100% inhibition rates against these contaminants. To determine the response of the micropropagated plantlets to AMF acclimatization, it was observed that Kiganda cultivar responded best to Rhizophagus irregularis which showed the highest recorded biomass at 0.97. There is need to emphasize collaborative efforts among various stakeholders in establishing sustainable practices. Additionally, low-cost TC is a viable tool for producing disease-free seedlings and intergrating antimicrobial agents is recommended to minimize contamination risks. Finally, the use of AMF treatment in acclimatization with further research needed for validation across different agroecological conditions. These recommendations aim to enhance productivity, sustainability, and resilience in banana farming systems.
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    Bioengineering Drought Tolerance in Tropical Maize (Zea Mays L.) through Inducible Expression of Isopentenyltransferase Gene
    (2014-08-26) Bedada, Leta Tulu
    Drought is one of the major abiotic constraints contributing to low productivity in maize. In tropical region, it causes grain yield losses of as high as 70% while complete crop failure is also common depending on the severity of drought. Drought diminishes crop productivity mainly by causing premature leaf senescence. It is now possible to delay drought induced leaf senescence in order to enhance tolerance to drought and stabilize crop yield through bioengineering. The ipt gene codes for isopentenyltransferase (IPT) enzyme, which catalyzes the rate- limiting step in the biosynthesis of cytokinin (CK) and enhances tolerance to drought by increasing the foliar level of CK that delays drought-induced leaf senescence in transgenic crops. This study was designed to genetically transform locally adapted elite and commercial tropical maize genotypes with ipt gene to develop drought stress tolerance through Agrobacterium tumefaciens-mediated genetic transformation. Ten maize genotypes adapted to Ethiopian and the Eastern and Central African (ECA) countries were evaluated for in vitro regeneration ability using immature zygotic embryos as explants. Six genotypes (Melkassa-2, Melkassa-6Q, [CML387/CML176]-B-B-2-3-2-B [QPM], CML395, CML442 and CML216) were identified as the best regenerating ones having potential for improvement through genetic transformation. Subsequently, the ipt gene was sub-cloned into the pNOV2819 binary vector to take advantage of the pmi gene as plant selectable marker and mannose as selective agent. The pNOV2819 binary vector carrying the ipt gene was introduced into the Agrobacterium strain EHA101 which was subsequently used to transform immature zygotic embryos obtained from the six genotypes. Among the six genotypes studied, transgenic plants were successfully regenerated in Melkassa-2 and CML216 with regeneration efficiency of 87.5 and 59.6%, respectively. Transgenic plants were analyzed using PCR, Southern blot and RT-PCR. Based on PCR results, transformation efficiencies were found to be 97.4 and 100% for Melkassa-2 and CML216, respectively, indicating stringency of the pmi/mannose based selection system for maize transformation. Southern blot analysis indicated stable integration of the transgene into the genome of CML216 with 2-3 copy numbers in five independent events. In drought assay carried out in the glasshouse transgenic plants expressing the ipt gene maintained higher leaf relative water content (RWC) and total chlorophyll concentration during the drought period and produced significantly higher grain yield, major yield components and root dry matter compared to the non-transgenic plants. The ipt gene was observed to improve drought tolerance in tropical maize by delaying drought induced leaf senescence. It was concluded that the transgenic line developed can be further tested for tolerance to drought under contained field trials. Furthermore it can be used in breeding programs to improve drought tolerance in other commercial tropical maize genotypes through conventional breeding.
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    Bovine interleukin-3: cloning production and gene expression in cattle infected with trypanosoma angolense
    (2012-05-04) Mwangi, Simon Musyoka
    Interleukin-3 (IL-3) is one of the cytokines that act during the early and late stages of blood cell formation. To enable the study of the role of IL-3 in bovine haemopoietic stem cell differentiation, the polymerase chain reaction (PCR) was used to amplify an IL-3 cDNA from first strand cDNAS's prepared peripheral blood mononuclear cells (PBMNC) from N'Dama and Boran cattle. An analysis of the cDNA sequence reveals that it contains a 432-nucleotide (nt) open reading frame which codes for 144 amino acids (aa) . Cleavage of the putative signal peptide consisting of the first 17 aa yields the mature form of the protein (14.5 kDa). Comparisons of the bovine IL-3 sequence shares 90.7, 55.8, and 51.9 percent nt identity, respectively, in the coding region, and 85.4, 35 and 27.7 percent aa identity, respectively. The availability in recombinant form of large quantities of the mature protein encoded by this cDNA has enabled studies on the role of the cytokine in bovine haemopoiesis. These studies have shown that bovine IL-3 is a multipotential colony stimulating factor (CSF) whose activities include stimulation of the in vitro formation from bone marrow cells of eosinopil, neutrophil and macrophage colonies and in the presence of erythropoietin, of burst forming unit-erythroid (BFU-E) and mixed colonies. The expression of IL-3 mRNA was evaluated by reverse transcriptase (RT PCR) in cells from several organs from healthy Boran and N'Dama cattle to determine the sites of IL-3 gene expression and to gain an understanding of how the expression of the gene is controlled in cattle. IL-3 mRNA could not be detected in unstimulated PBMNC, unsorted bone marrow mononuclear cells (BMMNC), BMMNC enriched for T lymphocytes, lymph node mononuclear cells (LNMNC) and pleen mononuclear cells (SPMNC). However, follwoing stimulation with ConA, large amounts of the transcript were detected in all the cells with the exception of unsorted BMMNC. Studies of the kinetic patterns of IL-3 mRNA accumulation in LNMNC and SPMNC revealed that the mRNA accumulates rapidly following ConA stimulation, peaks after 3-6 h of stimulation and thereafter declines. To determine if there are any shared regulatory elements between the bovine and human IL-3 genes that might explain the similar patterns of gene expression observed in this study, a DNA containing a portion of the 5' flanking region of the bovine IL-3 gene was amplified by PCR from a bovine genomic DNA library. A search for homology with known IL-3 sequences in the GenBank database reveals that the first 487 nt of this DNA share 93.3, 66.5, 65.6 and 57.4 perent identity, respectively, with the sequences of the 5' flanking regions of the ovine, human, Rhesus monkey and murine IL-3 genes. A computer-assisted analysis of the sequence reveals several potential regulatory motifs, which closely match regulatory elements of the promoter of the human IL-3 gene both in their nt composition and spatial distribution. The further characterization of these DNA motifs will contribute to the understanding of how the expression of the IL-3 gene is regulated cattle. The expression of IL-3 mRNA in LNMNC, PBMNC and SPMNC from Trypanosoma congolense-infected cattle was also investigated to determine if depressed IL-3 gene expression plays a role in the ineffective haemopoiesis observed during infections of cattle with the African trypanosomes. In both Boran and N'Dama cattle infected with Trypanosoma congolense, IL-3 mRNA expression was never detected in unstimulated cells at all times of assay. Following stimulation of the cells with ConA, the amounts of IL-3 mRNA induced in LNMNC and PBMNC from the infected cattle were equal to those induced in cells from the non-infected controls. However, the amounts induced in SPMNC from infected cattle were significantly lower than those induced in cells from control animals. In summary, results of these studies demonstrate that bovine IL-3 has similar biological activities as IL-3 from other species, and suggest that bovine IL-3 is an important growth factor in induced haemopoiesis in cattle. They also show that, although the capacity of bovine T lymphocytes to express the IL-3 gene might not be impaired at the cellular level during infections of cattle with T. congolense, overall the production of IL-3 might be affected due to reductions in IL-3 gene expression in the spleen initially, and possible in other lymphoid organs in long-term trypanosome infections.
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    Cancer Risks Associated with Exposure to Background Ionizing Radiation in Human Habitat, Soil, and Food in Kenya
    (kenyatta university, 2023-10) Ogola, Phillip Einstein; Richard Okoth Oduor; Mathew Piero Ngugi; Siphila Mumenya Wanjiku
    Cancer is described as uncontrollable cell proliferation in the body. By spreading outside their borders and infiltrating neighbouring tissue, such cells can invade to other organs. Cancer is the third leading cause of mortality in Kenya, trailing only infectious and cardiovascular diseases, with an annual incidence increasing from 37,000 new cases in 2012 to 48,000 new cases in 2018. Similarly, annual cancer mortality increased from around 28,000 to 33,000 within the same time span. Cervical, breast, colorectal, prostate, and esophageal cancers are the most prevalent types of cancer in Kenya. According to the National Cancer Control Strategy 2011-2016, cancer arises from abnormal alterations resulting from interactions between genetic and environmental elements, which encompass physical carcinogens such as ionizing radiation. The GLOBOCAN 2020 estimates reported that in Kenya, there were 42,116 new cancer cases and 27,092 deaths attributed to cancer. Types of cancer linked to high radiation dose exposure include leukemia, breast, bladder, colon, liver, lung, esophagus, ovarian, multiple myeloma, and stomach cancers. Naturally Occurring Radioactive Materials (NORMs) are found in trace amounts in soil, water, food, and the atmosphere. While natural radioactivity is formed from primordial radionuclides spread throughout the earth's crust, human activities such as nuclear power generation and mining contribute to the release of additional radionuclides into the environment. Human exposure to radiation comes through external sources as well as consuming radionuclides through water and food ingestion or breathing of radioactive vapors. This research focused on establishing the cancer risks associated with exposure to background ionizing radiation in quarries, human premises, soils from agricultural zones, and tuber crops (cassava, sweet potatoes, and Irish potatoes) in Kisii, Kakamega and Busia Counties in Kenya. Ionizing radiation levels were detected using handheld radiation detectors in quarry sites and buildings, whereas 232Th, 40K, and 238U activity concentrations in soil and tuber crops were determined using high purity germanium detectors. In quarry sites, the calculated average annual effective dose (AED) was 1.702 mSv in Kisii County, 2.083 mSv in Kakamega County, and 2.016 mSv in Busia County. Furthermore, the mean excess lifetime cancer risk (ELCR) due to occupational exposure in quarry sites were 5.702×10-3 in Kisii County, 6.979×10-3 in Kakamega County, and 6.754×10- 3 in Busia County. The computed AED values in the premises were 1.369 mSv in Kisii County, 1.317 mSv in Kakamega County and 1.268 mSv in Busia County. Also, the lifetime cancer risk attributable to indoor background ionizing radiation exposure were 4.586×10-3 in Kisii County, 4.411×10-3 in Kakamega County and 4.247×10-3 in Busia County. From the findings, cancer risk associated with lifetime exposure to ionizing radiation in quarry sites and premises sampled herein was low. Moreover, the mean activity concentrations of 232Th, 238U, and 40K in soil samples were 83.58 Bq/kg, 28.6 Bq/kg, and 483.40 Bq/kg respectively in Kakamega County, 65.36 Bq/kg, 22.88 Bq/kg, and 176.72 Bq/kg respectively in Busia County and 106.6 Bq/kg, 46.08 Bq/kg, and 153.42 Bq/kg respectively in Kisii County. Additionaly, the mean activity concentrations of 232Th, 238U, and 40K in tuber crops sampled in this study were below the global average values. Furthermore, the estimated mean annual ingestion effective dose (AIED) values in tuber crops ranged from 0.049 mSv to 0.062 mSv for cassava, 0.027 mSv to 0.064 mSv for sweet potatoes, and 0.057 mSv to 0.153 mSv for Irish potato. Similarly, the computed radiation hazard indices in soil samples and tuber crops yielded values below the acceptable limits, indicating not only insignificant cancer risk associated with the general public's exposure to ionizing radiation from the soil samples but also safety in consumption of these tuber crops
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    The causes and effects of pica habit of pregnant women in Msambweni division, Kwale District, coast province
    (2011-08-12) Kinyua, Felix
    Pica, the habit of eating non-food substances is a worldwide behaviour that is not limited to any geographic area, sex or status. It is common in retarded children and adults with some forms of insanity, hysteria and pregnancy. Geophagia is a form of pica where individuals consume soil and its products. Though the aetiology of Geophagia is not well elucidated, it could result more from habit, culture and superstition than from need for specific nutrients. Investigations to establish a cultural link to Geophagia among the Digo community revealed that 28% of women gave clay to their young children who wanted to taste what the mother ate. Thereafter, children ate clay as food. In clinical practice Geophagia is thought to occur due to iron deficiency anaemia. To establish the causes and effects of the habit in adults, 236 women were recruited for the study. 182 were pregnant geophageous and non geophageous test subjects. 54 were non-pregnant geophageous and non geophageous comparative study subjects. Through a questionnaire, commonly used substances and reasons for ingestion were investigated. The most common substance in the study area was found to be a synthetic product which is imported from Egypt, India and Singapore either as Portland cement or basmati rice and locally sold as roasted clay with herbs and spices. The substance is highly addictive and after long use, it kills appetite for other substances except itself. Investigations for anaemia showed that the substance is associated with high Hb, PCV and MCHC values that subsequently decline leading to chronic anaemia. However, anaemia even with active worm infestation was not able to cause Geophagia. Microbial analysis of the samples indicated contamination with air and faecal-borne bacteria including B.subtilis, P.mirabilis and Str. faecalis. Anti-microbial susceptibility tests showed various reactions to different drugs. Analysis of the trace element profile for local and imported soil substances indicated the presence of high levels of essential and radioactive elements which exposed photographic film. The latter elements include titanium, rubidium, strontium, zirconium, and niobium. The classic radio-nuclides that include uranium-238, thorium-232 and potassium-40 were also detected in significant quantities. All results were shown in text, tables and histograms where applicable
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    Changes in Serum Levels of Haematological and Biochemical Parameters, Erythropoietin and Pro-Inflammatory Cytokines in HIV-Infected Patients at Kenyatta National Hospital-Kenya
    (Kenyatta University, 2021) Mrama, Jackson Ireri; Joseph J.N, Ngeranwa; David N, Mburu; Walter Jaoko; Christine Sekadde-Kigondu
    There were between 31.6 million and 44.5 million HIV infected people globally by the year 2019, 70% of them in sub-Saharan Africa and Kenya harbored 1.6 million. Anaemia is the most common complications in HIV. Erythropoietin, and the proinflammatory cytokines (TNF-α, IL-6 and CRP) are markers of anaemia. The liver produce CRP and the kidney Epo. Changes in blood cell morphologies, levels of haematological, biochemical parameters, Epo, the proinflammatory cytokines in HIV have not been determined among Kenyans. This study aims at determining changes in the levels of these parameters in HIV infected patients. A total of 184 HIV infected adults seen at KNH and 202 blood donors formed the basis of this study. The HIV infected were grouped into: CD4<200 -, 200-499- and ≥500 cells/mm3 and followed for 6 months. Blood samples for assessing levels of the parameters were taken. SPSS version 21 was used to analyze the data, Shapiro-Wilk test determined the normality of the data. Bootstrap parametric and non-parametric methods were used to raise the power of the low sample sizes of referents. Data was described by medians, 2.5-97.5th centiles, means and standard deviations. Wilcoxon rank-sum test compared independent variables and Kruskal Wallis test compared values between CD4 groups. Among the referents, significantly higher mean values in females than in males were demonstrated in: WBC (p=0.002), N (p =0.004), L (p=0.001), CD4+ cells (p<0.001) and P (p<0.001); while lower mean values were demonstrated in RBC (p<0.001), HB (p<0.001) and E (p=0.004). In Biochemical values, females demonstrated significantly higher mean values than males in ALB (p<0.001), but lower in AST (p=0.001), ALT (p<0.001), ALP (p< 0.001, T.Bil (p=0.004), D.Bil (p<0.001), T.PRT (p<0.001) and CRT (p=0.001). In cytokines significantly higher median values in females were demonstrated in CRP (p<0.00I) and lower in: Epo (p<0. 020), TNF-α (p<0.001) and IL– 6 (p=0.016). During follow up, microcytic hypochromic anaemia coupled with rouleaux of up to 15.4% and 15.4% were demonstrated in ART and ARV – naïve females and up to 50% in ART males. ART females demonstrated significantly higher RBC and HB mean values than ARV females. Macrocytic anaemias together with targets of up to 28.2 %, 50% in ART and ARV-naïve females respectively and up to 25% in both ART and ARV-naïve males were demonstrated. Neutrophilia together with hypersegmentation of up to 15%, 25% in ART and ARV-naïve females respectively and 25% in males; eosinophilia of up to 11.1% in ART females thrombocytosis of up 11.1% in ART females and 25% in males in CD4 ≥ 500 group; Lymphopenia of up to 33% in ART females and 50% in ARV males decreasing with increases in CD4 counts were demonstrated. In all the CD4 groups of all the respondents increases in AST, ALT, and GGT were demonstrated. Increases in ALB and ALP were demonstrated in ARV-naïve females in CD4 < 500 group. Decreases in T.PRT and increases ALB were demonstrated in females. Demonstrated were baseline increases in Epo, TNF-α and decreases in CRP and IL-6 and persistent increase in TNF-α in ART females. Recommendations were: development of gender-based reference ranges for: routine Haematological and Biochemical pararmeters, proinflammatory cytokines and use of L counts in HIV management in resource-limited setting.
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    Cloning Abiotic Stress Associated Genes and Agrobacterium tumefaciens Mediated Transformation of Selected Tropical Maize
    (2014-02-18) Abdalla, Rasha Adam Omer
    Drought and salinity are the main abiotic constraints of maize production. The genes confer tolerance to abiotic stresses can be isolated, cloned and introduced into important crops such as maize. The Annexin1 (AnnAt1) and Annexinp35 have been highly associated with water stress tolerance. AnnAt1 is induced by various stresses including water and oxidative stress and has been found to have peroxidase activity. Annexin p35 is a maize gene that also has peroxidase activity and plays a role in exocytosis, calcium transport and regulation. The Na+/H+ antiporter (NHX1) gene is involved in compartmentalization of Na+ into the vacuoles and therefore playing an important role in salt tolerance. However these genes have not been reported in transformation of tropical maize and its response to water stress. Annexinp35 has never been cloned and used in transformation of any crop. The objectives of this study was to isolate and clone the maize Annexinp35 gene and transform tropical maize with the Annexinp35, AnnAt1 and NHX1 genes conferring drought and salt tolerance via Agrobacterium tumefaciens and screening for stable integration and expression of the transgenes in T0 transgenic plants. An initial assessment of maize response to callus induction was performed using mature embryos, shoot tips and leaf segments and different levels of 2,4-Dichlorophenoxyacetic acid (2,4-D). AnnexinP35 gene was isolated from egg cells of maize and cloned into pNOV2819 vector, the AnnAt1 gene in pROK2 vector was sub-cloned into pNOV2819 vectors with it is promoter and terminator, the PMI gene from pNOV2819 vector was sub-cloned in to pCAMBIA-NHX1 vector which carries the salt tolerant gene NHX1 gene. Drought and salt tolerant genes were engineered into Sudanese maize genotypes, These genotypes are both striger resistant and most preferred by farmers in Sudan. The drought and salt tolerance genes were introduced to Sudanese maize using Agrobacterium tumefaciens method. The experiment was carried out using random complete block design. Transformation frequency and efficiency were assessed by using mannose as selectable agent. Transformation frequency and efficiency were found to be genotypic dependant. Transformation frequency and efficiency were evaluated for all the genotypes used in the study. Shoot tips gave the highest callus induction frequency among all the genotypes used while mature embryos gave the lowest callus induction frequency. The highest transformation frequency for the gene construct pCAMBIA-NHX1/PMI was observed in STR136 (8.03%). IL15 and Mojatamma-45 had TF of 2.05% and 1.75% respectively while the highest TE was observed in IL15 (1.13%). The highest transformation frequency for the vector pNOV2819- ASARZMANNp35 was observed in Hudiba-2 (31.78%) while the lowest TF was observed in Mojtamma-45 (5.38%). The highest TE was observed for Giza-2 (1.00%). The gene construct pNOV2819-AnnAt1 gave highest transformation frequency in the Inbred local-5 (23.98%) and lowest TF in IL1 (3.75%). The highest TE was observed Giza-2 (2.48%) for the same gene construct. Drought tolerant lines generated will be available to the maize breeders to transfer the trait to lines that have high yield but lack this trait.
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    Cognitive Enhancing Effects and Antioxidant Activities of Crude Methanol Extracts of Prunus Africana in Scopolamine-Induced Alzheimer’s Disease Mice
    (Kenyatta University, 2023) Ngai, David Nyaga; Mathew Piero Ngugi; Cromwell Mwiti Kibiti
    Alzheimer’s disease (AD) is a type of dementia marked by gradual progressive neurodegeneration caused by excessive neurofibrillary tangles and amyloid beta (Aβ) build-up in hippocampus and other parts of the brain. Ultimately, this reduces the number of neurons, synapses and pyramidal nerve connections resulting in compromised cognitive functions and memory loss. The immune system clears senile plaques and neurofibrilary tangles oxidatively and in the process, there is excessive generation of reactive nitrogen and oxygen species that cause the chronic oxidative stress observed in AD. Conventionally, cholinergic agonists, particularly inhibitors of acetylcholinesterase including rivastigmine, donepezil and galantamine are the most commonly used drugs in AD therapy. Unfortunately, their efficacy is low and they have short half lives hence higher and more frequent dose requirement which is limited by their side effects that are sometimes unbearable. Fortunately, there are phytochemicals with AD ameliorating effects. These include phenolic compounds that clear aggregated amyloid proteins and have antioxidant and anti-inflammatory properties, steroidal alkaloids that have anti-cholinesterase activity while ascorbic acid, terpenoids and rivastigmine are not only free radical scavengers but also stabilizers of muscarinic receptors. Antioxidant activity is a vital requirement for plant extracts that are used with great success to manage degenerative diseases like AD since such diseases are usually accompanied by oxidative stress. In the current study, in vivo cognitive advancing and antioxidant effects of crude methanol extract of stem bark and leaf of Prunus africana were investigated in scopolamine-induced AD mice. Morris water maze and Passive avoidance task were used to measure spatial memory and cognitive enhancing effects of the two plant extracts, respectively. Scopolamine butylbromide (5mg/kg bw) was administered intraperitoneally to induce AD during the study. Donepezil (DNP) was used as the standard drug in the current study because of its ability to improve and maintain high global cognitive potentials and high torelability. The drug substance, scopolamine, is the most commonly used substance in biosreening for AD drugs. It is known to lower transcription of brain derived neurotrophic factor responsible for memory elasticity and development and also interferes with functioning of muscarinic receptors. The study revealed phytocompounds with anticholinesterase activity including chlorogenic acid, pCoumaric acid, vitexin, isovitexin, sitostenone and campesterol. Phytocompounds with enzymatic antioxidant activity including ursolic acid, campesterol and sitostenone were also revealed. The two extracts also proved to be rich in phytochemicals with antioxidant and anti-inflammatory activities which are important neuroprotective properties. The two extracts significantly improved cognition and spatial memory in the two behavioural experiments. This was indicated by significant increase in escape latencies in passive avoidance task and decrease in escape latencies in Morris water maze tasks in a dose related pattern. These behavioural results are in collaboration with the anticholisterase, antioxidant and anti-inflammatory activities of compounds revealed in the two extracts. Generally, the stem bark extract had significantly higher efficacy than leaf extract in most of the tests carried out. The disparity in efficacies of the two extracts can be explained in terms of differences in diversity and proportions of phytochemicals present. From the current study, it is concluded that crude methanol extracts of stem bark and leaf of P. africana have several phytocompounds with cognitive and spatial memory enhancing effects, anticholinesterase and antioxidant activities in scopolamine induced-Alzheimer’s disease mice.
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    Determination of CD8+-cell responses in a high risk HIV negative population
    (2011-08-23) Muturi, Margaret Wangui
    Naturally acquired cellular immunity in individuals who have been exposed to HIV-1 but have remained uninfected may hold clues for the design of an effective HIV vaccine. IFNy Elispot has emerged as one of the widely used assay to monitor HIV-specific immune responses. It is becoming the assay of choice for evaluation of HIV-vaccine-induced cell-mediated immune responses in many clinical trials. The objective of this study was to investigate the CTL responses of high risk HIV seronegative individuals to HIV A and RENTA vaccine peptides. The study further sought to investigate whether it was possible to recruit, sample, counsel and follow-up a cohort of high risk seronegative volunteers over a duration time in preparation for vaccine trials. To achieve these objectives, 30 volunteers filled a questionnaire, were counseled, tested for HIV status, recruited and enrolled in a 15 month study.The thirty exposed seronegative (ES) volunteers reported frequent unprotected sex with people of unknown HIV-1 status at enrollment. Every 3 months the volunteers were seen at the KAVI Kangemi clinic where blood samples were taken for the determination of the CTL responses, their HIV status was re-checked, filled questionnaire to assess the changes in their risky sexual behaviour. It was possible to recruit and follow-up the 30 volunteers for the entire duration of the study. All the thirty samples did not show HIV-1 specific T cell responses to both RENTA and HIV-A peptides using the ex vivo Elispot assay during the four time points (months 0, 3, 6 and 9). To investigate whether these results were truly negative, samples from 5 seronegative discordant couples were used. There were no HIV-1 specific CD8+ IFNy T cell responses in the HIV negative spouse. To investigate whether the ex vivo Elispot was unable to detect the responses, cultured Elispot assay was applied to the samples. They all tested positive with variations between peptide pools and individuals. The fact that cultured Elispot detected the responses from the 5 seronegative spouses of HIV infected partners and from 12 of the thirty means that the ex vivo Elispot assay was not sensitive enough to detect responses to the tested vaccine peptides. Cultured Elispot expands the memory CTL thus enhancing the detection of the responses. Using this method it was possible to demonstrate that HIV-1 specific CD8+ IFNy T cell responses exist in high risk exposed seronegative individuals. Pool 90 gave positive responses with all the samples. It would appear that combining the pools of peptides would elicit consistent CD8+ IFNy T cell responses and therefore make a better vaccine candidate. The results suggest that there is need to exercise very stringent criteria for enrolling high risk exposed seronegative participants to any study group meant to investigate immunological parameters related to HIV exposure.
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    Determination of Wilt Disease Resistance, Performance and Environmental Safety of Transgenic Bananas Expressing Hrap-Pflp and Espflp Genes under Field Conditions
    (Kenyatta University, 2024-04) Muchira, Dancun King’ang’i
    Banana Xanthomonas wilt (BXW) disease is caused by the bacterium Xanthomonas campestris pv musaceae. Transgenic banana expressing the genes hypersensitive reactive associated protein (Hrap), plant ferredoxins-like protein (Pflp), and extracellular secreted plant ferredoxin-like protein (EsPflp) have been developed and shown to control BXW in glasshouse in Uganda. However, the transformed banana lines have not been evaluated in Kenya for BXW disease resistance, field performance, and environmental safety. BXW disease causes over 50% losses of production in farmer’s fields. Banana production is essential to Kenya and the region as it provides food, fodder, and income. In Uganda, these transgenes have been used to transform banana cultivars of Sukali Ndiizi (AB) and found to be resistant to BXW under glasshouse and field trials. Non-target effect studies of the transgenes have also been conducted in Uganda for non-target soil bacteria colonization. As demonstrated in the trials, the technology has potential, so conducting field trials for the transgenic banana events in Kenya is important to generate data on disease resistance, performance and environmental safety before they are incorporated into agricultural production. With respect to this, a confined field trial was established in Busia County at Kenya agricultural and livestock research organization (KALRO) Alupe research station. Randomized complete block design (RCBD) with 30 banana events replicated 12 times was used. Bananas were planted at a spacing of 3m by 3m from each other and a hole size of 2.5 cubic feet. During planting, 20 kg of well-composted manure and 100 g of double ammonium phosphate (DAP) were used for each banana stool. At 5 months after planting, molecular characterization of the plants was carried out. The plants were then physically infected with the wilt-causing cultures. Disease resistance was carried out by scouting for disease symptoms post-infection. Morphological/Agronomical data was collected on traits such as height, girth, and leaf total area. Soil studies were also conducted by analyzing plant litter decomposition rate and carbon readings. Environmental studies were conducted by analyzing the numbers of soil nematodes, flying insects, and banana weevils. All the data were analyzed using one-way ANOVA, and significance levels were read at α=0.05. In the BXW disease resistance evaluation and agronomy, the effects of the transgenes could not be determined, as the study ended prematurely because both transgenic and non-transgenic plants died within 12 months. The differences between transgenic and non-transgenic control in soil qualities, soil nematodes, flying insects, and banana weevils did not differ significantly (p˃0.05). The study implies that Hrap-Pflp and EsPflp are environmentally safe as they did not affect non-target organisms and soil quality. The transgenes can, therefore, be selected for multilocation assessment to collect more data on safety.
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    Development of a genetic linkage map and quantitative trait loci (QTL) analysis in cowpea (vigna unguiculata (L.) walp)
    (2010-06) Andargie, Mebeaselassie; Muluvi, Geoffrey M.; Pasquet, Remy S.
    Cowpea is a diploid plant species which contributes significantly to food security in developing countries, especially in Africa. This research project was carried out in view of the upcoming introduction of Bt cowpea in Africa which is likely to alter the equilibrium existing within the cowpea taxa. The objectives of this study were to develop viable microsatellite markers and construct the SSR based linkage map, identify quantitative trait loci that regulate yield, domestication related traits as well as flower scent and identify the volatile compounds that attract pollinators to cowpea flowers. In order to achieve these goals 159 F7 recombinant inbred lines including the two parents and 206 markers (202 SSRs and 4 morphological) were used. The first SSR based linkage map of cowpea was constructed that spans a genetic distance of2991cM. QTL for seed weight (SW), domestication related traits (DRT), flower scent/aroma were mapped in all 159 F7 plants and the two parents 524B x 219-01. Six QTL associated with 74 % of the phenotypic variance were detected for SW on chromosomes 1, 2, 3 and 10. Both the 524B and 219-01 alleles increased SWat six of the QTL on chromosomes 1, 2, 3 and 10. For domestication related traits, nine QTL (four for testa size and five for pod fiber thickness layer) explaining 54.5 and 47.9 % of the phenotypic variance, respectively were on chromosomes 1,2, 4, 6, 7 and 10. The 524B allele increased DRTs at three-fourth of all QTL. QTL for SW and DRTs were clustered on chromosomes 1 and 10. Association of SW and DRTs QTL may be the cause of the significant phenotype and genotypic correlation detected between the two traits. The test of linkage vs pleiotropy for SW and DRT QTL on chromosomes 1 and 10 suggested pleiotropy. For flower scent/aroma, 63 QTL were detected on chromosomes 1,2,3,4, 5, 6, 7, 8, and 10. In addition, a total of twenty-two different volatiles were identified by the GS-MS technique. Clustering ofQTL were observed on chromosomes 1,2, and 4 mainly, suggesting that it can occur either due to the presence of a single locus with pleiotropic effects on several volatiles or as a result of tightly linked different loci. Such loci may encode transcription factors that co-ordinately regulate genes, or they may encode enzymes that catalyse limiting steps in single pathways. It is anticipated that this resource will have an important impact towards the development of marker assisted selection systems for the cowpea breeding community, and for future genetic studies in cowpea.
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    Development of Rapid Diagnostic Tool and Characterization of Rift Valley Fever Virus Infection in Kenya
    (Kenyatta University, 2024-06) Wekesa, Francis Chaka
    Rift Valley fever (RVF) is a high-priority zoonotic disease characterized by massive loss of livestock within a short period of its outbreak. The disease is endemic in sub-Saharan Africa and mainly spread via infected female Aedes and Culex genera of mosquitoes. This study aimed to develop a rapid diagnostic tool and spatio-temporal characterization of RVF in Kenya. During this study, a calorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of Rift Valley Fever Virus was developed and validated. In addition, RVF infection was characterized through generation of the current RVFV transmission risk maps of Kenya by computation of the basic reproduction number (Ro) based on temperature change, presence of vector and host, and the host recovery rate. The risk maps were validated using a serological study that was carried out on 615 blood samples collected from Siaya, Busia, and Kisumu counties in the year 2018. The RVFV IgG and IgM antibodies were screened and the results confirmed using either serum viral neutralization test and/or real time polymerase chain reaction (RT-qPCR). The novel diagnostic tool that was developed detected the presence of the virus within 30 minutes at a constant temperature of 65oC with a sensitivity of 98.36% and a specificity of 96.49%. The RVFV transmission risk maps showed the likelihood of having outbreaks in seventeen counties of Kenya. Most of these counties were from arid and semi-arid lands (ASALS) whose livelihood greatly depends on livestock. However, the possibility of the disease to occur outside ASAL areas was demonstrated by the presence of IgG and IgM antibodies in counties previously perceived as RVFV-free zones. The average seropositivity rate of RVFV IgM antibodies in the three counties was estimated to be 10.33% with Siaya county leading with 24% (95% CI (2.269-8.009) OR 4.26) followed by Busia County 7% (95% CI (0.532-1.88) OR 4.26). Kisumu County recorded Zero RVFV positivity rate. The overall seropositivity rate for RVFV IgG antibodies from the three counties was at 14% with Siaya County leading at a positivity rate of 22% (95% CI 0.805 -2.140) OR 1.31), followed by Busia 18% (95% CI 0.349 -2.867) OR 1) and Kisumu 2% (95% CI (0.032-0.265) OR 0.09). The colorimetric-RVFV-UDG-RT LAMP assay was ten-fold more sensitive compared to the RT–qPCR. This is attributed to many start points for amplification in the LAMP technology as opposed to single start point in real time polymerase chain reaction (RT–qPCR). The RVF spatio-temporal model correctly predicted regions and months that the disease is likely to occur. The presence of undetected circulation of RVFV IgG and IgM antibodies in Siaya, Busia, and Kisumu counties, points to a possible impending future outbreak of the disease in these areas as a result of the paradigm shift in weather conditions due to climate change. Siaya county showed highest prevalence of the RVFV followed by Busia county due to their proximity to Uganda that recently experienced RVF infection outbreak in one of the districts. Therefore, the kit can be adopted and deployed for rapid screening of RVFV in Kenya.This creates the need to focus and invest in the RVF infection control measures. The findings of this study calls for increased disease surveillance to cover wider western borderline counties and proper emergency preparedness in case of RVF outbreak so that the disease is managed on time to minimize losses during outbreaks. More importantly, RVF risk maps could contribute to data-driven decision-making for disease management when deploying vector control interventions.
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    Diagnosis and Epidemiology of Zoonotic Nontuberculous Mycobacteria among Dromedary Camels and Household Members in Samburu County, Kenya
    (Kenyatta University, 2020) Lucas, Luvai Azaale Asaava
    Zoonotic nontuberculous Mycobacteria (NTM) cross infect a wide range of domestic animals, wildlife and man causing various diseases. Despite the public health implications associated with mycobacterial infection and the existing close interaction of pastoralists and camels, information on diagnosis and epidemiology of zoonotic NTM amongst camels and closely associated community members is scanty. The present study was a one-health approach study involving diagnosis and epidemiology of zoonotic NTM infection among dromedary camels and associated household members in Samburu East sub-county. The study was cross sectional covering camels slaughtered at county abattoirs as well as household members and camels. Abattoirs sampled included Isiolo and Athi-River camel abattoirs, where all sampled camels were confirmed to have come from Samburu east. Household camel sampling strategy involved stratification of the study area to Wamba and Waso rearing areas. Sampling at abattoirs was consecutive for camels identified to have TB-like lesions during meat inspection. People presumed to have TB from amongst the sampled households were requested to provide a sputum sample. Screening of lactating camels for Mycobacteria infection was done using single intradermal comparative tuberculin test (SICTT) in a consecutive sample of 612 lactating camels from 83 households. A consolidated milk sample from all four mammary quarters was collected from 238 tuberculin test reactive camels. Post mortem (PM) examination was conducted on 1600 camels originating from the study area. A semistructured questionnaire was administered to collect data on risk factors from 83 respondents. All milk, sputum and tissue samples were analysed at Kenya Medical Research Institute (KEMRI)/Centre for Respiratory Disease Research (CRDR) enhanced BSL2 laboratory using mycobacteriology, molecular speciation using GenoType® Mycobacterium line probe assay (HAINLifescience), drug susceptibility testing, 16S rDNA sequencing and phylogenetic analysis. This study was conducted between April, 2017 and December, 2018. Results indicated that the proportion of M. bovis and M. avium reactors was estimated at 6.05% and 39.38% respectively. Out of 238 milk samples, 57 (23.95%) had culture positive acid fast bacilli (AFB). Out of 132 suspected lesions on PM, 27/1600 1.69% (binomial 95%, CI: 1.11%-2.45%) were AFB positive on culture. Of the 48 sputum samples, 7 were AFB culture positive. The NTMs were observed to occur widely in various samples analysed, including post mortem lesions: M. fortuitum 17/27 (62.96%), M. scrofulaceum 3/27 (11%), M. szulgai 2/27 (7.4%); camel milk samples: M. szulgai 20/57 (35.09%), M. monacense 5/57 (8.77%), M. litorale 4/57 (7.02%), M. fortuitum 3/57 (5.26%), M. lehmannii 3/57 (5.26%), M. elephantis 3/57 (5.26%), M. duvalii 3/57 (5.26%); and in Human sputum samples: M. fortuitum 1/48 (2.08%), M. szulgai 2/48 (4.16%), and M. litorale 1/48 (2.08%) among others. These findings demonstrate high levels of infection with NTM in both humans and camels. Camel post mortem analyses revealed bronchial, mediastinal lymph nodes and lung lobes were most affected tissues. Several NTMs of medical importance with varying level of commonly used TB drugs sensitivity. The pattern of resistance to first-line TB drugs was as follows: Isoniazid (100%), rifampicin (97.9%) and ethambutol (65.3%). All NTM species isolated were highly resistant to first-line TB drugs. Camel breed, age, production system, origin of new introductions, migration and herd size were identified as risk factors for infection in camels (p<0.05). Surveillance and notification systems for NTMs including specific immunological test for NTMs diagnoses are needed. The public health significance of NTM in camels and humans needs further unravelling.
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    Distribution importance and management of stemborers (lepidoptera) in maize production systems of semi-arid Eastern Kenya with emphasis on biological control
    (2012-04-05) Songa, J. M.
    The bulk of maize, which is the main staple food in semi-arid Eastern Kenya, is grown by subsistence small-scale farmers, and the yields are usually low, averaging 500kg/ha Stemborers are one of the major causes of these low yields. The general goal of this project was to generate basic information for the development of a sustainable biological control programme for stemborers in this region, with emphasis on parasitoids. In a survey conducted in six agroecological zones (AEZs) in March-June, 1996, the pests reported to damage maize were: chafer grubs, stemborers, termites, 'red ants', yellow necked spur fowls, ground squirrels, monkeys, porcupines, rats wild pigs and storage insect pests. Squirrels were considered to be the most widely distributed and important vertebrate pest of maize in all the study zones. Stemborers were reported as pests of maize in all the zones, and ranked first among insect pests in AEZs: UM4, LM4, LM3, LM4 and second in LM5, but were considered unimportant in UM2. Agronomic practices that may influence stemborer infestation in maize, including cropping systems, varieties, sowing time, fertilizer/manure use, stover storage, usage and disposal are discussed. Farmers use insecticides, wood ash, soil, saw dust, chilli pepper, dry cell powder and Tagetes minuta to control stemborers. The spatial and temporal distribution of stemborers, and the incidence and severity of stemborer infestation in maize, was investigated in farmers' fields in six AEZs during the LR 1996 and SR 1996/97. The stemborers that infested maize in the six AEZs were: Chilo partellus, Sesamia calamistis, Cryptophlebia leucotreta and Busseola fusca. C. partellus was the most abundant and widely distributed, was most prevalent in the lower altitude zones, whereas S. calamistis and C. leucotreta were more common in the higher altitude zones. C. partellus was mainly responsible for the early to late season infestation of maize in the LM3-5 zones, while C. leucotreta and S. calamistis infested the crop mostly from mid-season especially in the UM2-4 zones. Stemborer infestion was much lower than expected for this region (1-22%), and was attributed tothe unusually low rainfall received during the two seasons. Stemborers were found to be a potential major problem to maize production in all the zones, except in UM2, which had the lowest severity of infestation (1.64). A study was conducted to determine the interrelationships among stemborer density, damage and plant growth variables, at the vegetative, reproductive and maturity stages and, their effect on grain yield in maize at Katumani, in the SR 1997/98 and LR 1998. Correlations and path coefficient analysis were used. The path analysis model, which accounted for 86% of the total variation in maize grain yield, showed that, the effects of larvae and damage variables, on the plant growth variables and grain yield, were primarily through damage caused at the vegetative stage. Among the plant growth variables, plant height at maturity, had the highest direct positive effect on grain yield. Among the damage variables, stem tunneling at the vegetative stage, had the higest indirect negative effect on grain yield, larvae at the vegetative stage caused a grain yield loss of about 3g per plant. Studies conducted at Katumani, Ithookwe and Kiboko, for four seasons (SR, 1996 - Lr 1998), revealed that, the stemborers that infested maize were C. partellus, S. calamistis, C. leucotreta and B. fusca, with C. partellus being the most abundant (71.3% - 92.89%) and widespread species, while B. fusca was the least common (0 - 0.12%). A complex of upto 22 different parasitoid species were recovered from the stemborers, and the parasitism was highest at Kiboko followed by Ithookwe and Katumani in descending order. The most common indigenous larval parasitoids were C. sesamiae and C. curvimaculatus, and the pupal ones were Pediobius furvus and Dentichasmias busseolae but their effect was low. During the season of introduction of the C. flavipes, (SR 1997) and the following (non-release) season, C. flavipes was responsible for the highest parasitism of stemborers in all zones (10.33% - 25.81%). This study showed that C. flavipes was able to locate, parasitize and successfully colonize stemborers (C. partellus, S. calamistis and C. leucotreta), at the three sites. Kiboko appeared to be the most suitable site for the survival of C. flavipes. Partial life table analysis of C. partellus showed that the highest mortality occurred on the 3rd and 4th instar larvae, and was mainly due to disappearance. However, key factor analysis showed that parasitism by C. flavipes on the 5th and 6th larval instars was the key factor that determined changes in the densities of C. partellus, during the period of this study. An on-farm study in the six AEZs from July, 1997 to April, 1998, showed that, Pennisetum purpureum grass was the most widespread and abundant wild host of C. partellus and S. calamistis throughout the study period, followed by Panicum maximum. C. partellus was the predominant species, with the highest number being recovered from S. versicolor (94.6%). Only larval parasitoids were recovered from C. partellus in S. versicolor, with C. flavipes causing the highest parasitism (13.64%).
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    Diversity of Moulds, Sources of Contamination, Moisture Content and Aflatoxin Levels in Fish and Fish Feeds in Kisii County, Kenya
    (Kenyatta University, 2024-02) Nyamwaka, Irene Seila
    Aflatoxins are toxic substances produced by some members of Aspergillus species which commonly invade foodstuffs as well as animal feeds. When consumed in quantities exceeding 10 µg/kg, they cause nephrotoxic and hepatotoxic effects in humans and other animals. Data on the levels of aflatoxins in fish and fish feeds sold in Kisii County is not available. This study was carried out to assess the diversity of moulds associated with fresh, sundried fish and fish feeds sold in selected markets in Kisii County, determine aflatoxin levels in sundried fish and fish feeds, evaluate the relationship between aflatoxin levels and moisture content in sundried fish and fish feeds and assess sources of fungal contamination of sundried and fresh fish sold in selected markets in Kisii County. Samples of sundried and fresh fish from retailers in four main markets in Kisii County were obtained by random sampling. Samples from fish farmers in Kisii County and shores of Lake Victoria were obtained to serve as control. Commercial and home-made fish feeds were purposively obtained from five main outlets and three self-help groups respectively. Moisture content of all samples was determined by oven drying and the levels of aflatoxin in sundried fish and fish feeds determined by ELISA. Isolated moulds were identified using macroscopic and microscopic characteristics and Sanger sequencing technique. Sources of fish contamination were determined using a structured questionnaire and level of contamination expressed as cfu/g. Data on aflatoxin levels and colony forming units in different types of fish and fish feed samples was analysed using one-way analysis of variance. The means that were significantly different were separated using Tukey’s HSD test. Two-way analysis of variance was used to analyse the interactions between markets and type of fungi, types of fish feeds and fungal species, markets and types of fish. Data from molecular work was analysed using MEGA version 4. Relationship between moisture content and aflatoxin levels in fish and fish feed samples was analysed using regression analysis. Thirty fungal species from fourteen genera namely; Acremonium, Alternaria, Aspergillus, Candida, Cladophialophora, Cladosporium, Cunninghamella, Fusarium, Microporum, Mucor, Penicillium, Phoma, Trichoderma and Verticillium species were isolated from fresh and sundried fish samples. Eighteen fungal species were isolated from commercial and home made fish feeds. They comprised of eleven genera; Alternaria, Aspergillus, Aureobasidium, Cladosporium, Eurotium, Fusarium, Mucor, Penicillium, Stachybotrys, Trichoderma and Rhizopus species. Aflatoxin levels in sundried fish ranged from 20-840 ng/kg and 80-500 ng/kg in fish feeds. The cfus/g from control samples were significantly low compared to those from fresh and dried fish samples (p < 0.05). Transportation, handling and storage were found to be sources of fish contamination. The relationship between moisture content and fish, moisture content and fish feed was statistically significant (R2 = 0.942, p = 0.000) and (R2 = 0.797, p = 0.000) respectively. The findings of this study offer new insights into the levels of aflatoxins and associated fungi in fish and fish feeds sold in Kisii County. Therefore, there is need for frequent surveillance on aflatoxin levels in dried fish and fish feeds sold in this County.