http://localhost/xmlui/handle/123456789/1280 2026-04-07T15:32:51Z http://localhost/xmlui/handle/123456789/6896 Assessment of Bacterial Diversity and Antibiotic Resistance Genes in Water Used by Eateries and Open Markets in Juja Omune, Alfrick Makori Water is an important bacterial habitat and a major avenue of dissemination of antibiotic resistant bacteria between and among different environments. Contamination of drinking water used in open market eateries with antibiotic resistant bacteria pose a direct threat to human health. This is because antimicrobial resistome monitoring in drinking water is not currently a routine standard inspection protocol of drinking water. This study aimed to investigate the occurrence of antibiotic resistance genes (ARGs) and variation in the composition of bacterial communities in drinking water collected from eighty-two sampling points in Juja. Water samples were collected using purposive sampling. Special media of MacConkey, Thioglycollate and Lauryl Tryptose Broth media were used for isolation of bacteria from the collected water samples. Culture and molecular techniques were used to profile the bacteria and detect antibiotic resistant bacteria (ARBs) and antibiotic resistant genes (ARGs) present in samples. Metagenomics sequencing and analysis was applied to investigate the ARB genetic profiles. Morphological, biochemical characterization, antimicrobial susceptibility and molecular detection of ARGs data (qualitative & quantitative) were analyzed using R software to generate heat maps (Manhattan metric) showing characteristic relationships and antibiotic susceptibility or resistance among bacterial isolates. QIIME (version 2021.4) software and DADA2 were used to analyze microbial profiles in collected water samples. Detection of antibiotic resistance genes data were analyzed using R software to generate heat maps highlighting their presence in water samples. Results showed abundance of bacterial phylum Proteobacteria (above 86%) across all the collected water samples from the open markets and eateries. The dominant phylum documented by this study belonged to Proteobacteria and comprised of; Wakulima Eateries (WE) (Acinetobacter 44.180%), Wakulima Open Markets (WOM) (Duganella 28.201%), Gachororo Eateries (GE) (Acinetobacter 15.189%), Gachororo Open Markets (GOM) (Acinetobacter 30.675%) Mwerevu Eateries (ME) (Acinetobacter 40.823%) and Mwerevu Open Markets (MOM) (Curvibacter 48.785%). This study reported the presence of antibiotic resistant priority pathogens from the WHO list; Priority 1 (Critical) where Pseudomonas spp detected in samples ME (12.163%) and WOM (0.323%), Acinetobacter baumannii detected in samples GE (2.975%) and WE (5.495%), Enterobacteriaceae detected in samples WE (0.125%), MOM (0.326%), WOM (0.875%), GE (0.452%) and ME (0.206%). The genus of Escherichia, Klebsiella, Raoultella and Enterobacter dominated in Enterobacteriaceae. Priority 2 (High) pathogen detected was the genus Staphylococcus in samples MOM (1.826%) and GE (0.962%). The ARGs of qnrD and sul2 were detected in all six samples (WOM, WE, GOM, GE, MOM and ME), int1 and FloR genes were present in five samples except in MOM and GOM samples respectively while those of strB (MOM), catA (MOM) and blaTEM (GOM) were found in one sample each using qualitative PCR assays. Findings of this study form a critical reference point for policy development and rapid mitigation strategies to prevent a possible outbreak of water-borne diseases in the area. Master of Science in Biotechnology 2026-02-16T00:00:00Z http://localhost/xmlui/handle/123456789/6766 Ecological Niche Modeling, Yield Impact Assessment and Improved Detection of SPFMV, SPCSV, and SPLCV in Sweetpotato in Kenya Wanjala, Bramwel Waswa Viral diseases in Kenya and sub-Saharan Africa cause significant crop losses. Over 30 viruses have been reported to infect sweetpotato. Sweet potato cholorotic stunt virus and Sweet potato feathery mottle virus are considered the most wide-spread and devastating. Sweetpotato begomoviruses (sweepoviruses) have increasingly been recognized as common in recent years. They cause no or show mild symptoms but result in 40 to 80% yield losses. This study aimed: i) To develop an ecological niche model for predicting the potential geographic distribution and risk areas of sweepoviruses in Kenya; ii) To assess the yield impact of begomoviruses and their interaction with sweetpotato feathery mottle virus and sweetpotato chlorotic stunt virus; iii) To validate a loop-mediated isothermal amplification assays for on-site detection of the main sweetpotato infecting viruses and iv) To evaluate tube array for the multiplex detection of sweetpotato viruses. The study developed an ecological niche model to predict the geographic distribution and risk areas of sweepoviruses in Kenya. Field surveys were conducted during 2017 and 2018 cropping seasons, targeting the main sweetpotato growing regions. Ecological niche models were constructed using MaxEnt software, incorporating bioclimatic variables and International Soil Reference and Information Centre (ISRIC) factors. MaxEnt consistently surpasses other methods in terms of predicting accuracy, and is highly user-friendly. Results showed high, medium, and low probability for sweepovirus persistence and spatial distribution in regions like Western, Nyanza, and South Rift. Central and Eastern regions showed medium suitability, while the coastal region was predicted to be medium to high risk. The model provided valuable insights for developing effective disease risk management strategies. The impact of Sweet potato leaf curl virus (SPLCV) infection on root yield in Kenyan sweetpotato varieties 'Kakamega' and 'Ejumula' was investigated. Results showed significant differences in yield loss from SPLCV infection, with 'Ejumula' suffering no significant loss, while 'Kakamega', more resistant to Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV), suffered an average of 47% loss. The results highlight the variability in sensitivity to SPLCV between sweetpotato cultivars and the lack of correlation between SPLCV-related symptoms and susceptibility to the virus. The study also highlighted the lack of correlation between resistance to RNA viruses SPCSV and SPFMV and DNA virus SPLCV. A simple and rapid loop‐mediated isothermal amplification (LAMP) assay for the detection of SPFMV, SPCSV and begomoviruses related to SPLCV (sweepoviruses) was developed. Laboratory validation recorded 100% diagnostic sensitivity for all three viruses. The LAMP assays were customized for field testing using a lyophilized thermostable isothermal master mix in a ready-to-use form that required no cold chain. The average time to positivity (TTP) was: SPFMV 5-30 mins, SPCSV 15-43 mins and begomoviruses 28-45 mins. LAMP on-site testing results were comparable to PCR and RT-PCR confirmatory laboratory tests. The LAMP assay is a powerful tool for rapid sweetpotato virus detection at a reasonable cost and thus could serve as quality control systems for planting materials. The operational performance of STAR (Sweet potato virus Tube-Array) for simultaneous detection of sweetpotato viruses was evaluated. STAR assay was compared with the conventional method Nitrocellulose Membrane Enzyme-Linked Immunosorbent Assay (NCM-ELISA), with 25 samples tested in parallel in Lima and Nairobi. Cohen's kappa index revealed a disagreement between the two assays, with STAR being more sensitive and detecting more viruses not detected by NCM-ELISA. Concordance analysis accessed by Bland-Altman plot and regression analysis results from the two labs agreed closely. STAR was found to be more sensitive and detected more viruses than NCM-ELISA. STAR's sensitivity, specificity, accuracy, and short turnaround time make it suitable for routine diagnosis, epidemiological studies, quarantine, and certification of the sweetpotato seed system. PhD in Biotechnology 2025-07-16T00:00:00Z http://localhost/xmlui/handle/123456789/6765 Development and comparison of a loop mediated isothermal amplification assay for the rapid diagnosis of lumpy skin disease Macharia, Ednah Serah Wanjiru Lumpy skin disease virus is a poxvirus in the genus Capripoxvirus and is closely related to sheeppox virus and goatpox virus. It’s economically important in cattle and a notifiable disease by World Organization for Animal Health. Lumpy skin disease (LSD) is endemic in most parts of Africa with small-scale farmers experiencing the highest loss during outbreaks due to restricted animal trade and costly control and eradication measures. Serological methods of LSD detection are sensitive, inexpensive but can be laborious and time-consuming while, molecular methods such as Polymerase chain reaction (PCR), and real-time PCR/quantitative PCR (qPCR) are sensitive but require expertise and sophisticated laboratories. Loop-mediated isothermal amplification (LAMP) molecular method is advantageous, as it does not require expertise or sophisticated equipment. Thisstudy aimed to develop a rapid, simple, specific, and sensitive detection method for LSD. Sixtytwo samples that included skin biopsies, whole blood, serum, and cell cultures were used. New LAMP primer (10_LSD) that could detect lumpy skin disease virus, was designed using Genome based LAMP primer designer (GLAPD) software. Samples were analyzed by LAMP assay and a gold standard (real-time PCR). A LAMP field-based extraction method using polyethylene glycol (PEG) was developed and used for the detection of lumpy skin disease virus. The 10_LSD had a kappa value of 0.32 against the qPCR gold standard. In terms of limit of detection, qPCR had a detection limit of 10-3 ng/µl while 10_LSD had a limit of detection of 1 ng/µl and. The 10_LSD assay showed sensitivity of 60% and a specificity of 86 %. The LAMP assay did not cross-react with closely related viruses like camelpox, Orf virus, and Pestes des Petit Ruminants but could amplify sheeppox virus and goatpox virus. The average time to positivity was 14-28 minutes. The study supports the adoption of the LAMP assay for rapid Capripoxvirus diagnosis as a simpler, effective, and rapid method of detection, monitoring, and controlling outbreaks and the spread of disease in a field set up. MSc Research Publication 2025-07-15T00:00:00Z http://localhost/xmlui/handle/123456789/6764 Development of Rapid Diagnostic Tests for the On-Site Detection of Capripoxvirus Macharia, Ednah Serah Wanjiru Capripoxviruses are responsible for significant transboundary diseases affecting cattle, sheep, and goats, and are notifiable diseases in Kenya due to their economic impact. Early and accurate detection is crucial for effective disease control and surveillance. Current serological assays, while affordable and sensitive, are time-consuming, and molecular techniques such as PCR and qPCR require specialized equipment and expertise. In contrast, loop-mediated isothermal amplification (LAMP) offers a rapid, cost-effective alternative that can be performed with minimal infrastructure.This study aimed to develop and validate a LAMP assay that is efficient, rapid (<30 min), specific, and sensitive for the detection of Capripoxviruses. The study work was a cross-sectional laboratory activity design over two years, 2018 and 2020. Sampling was based on a non-probability sampling technique where convenience sampling was employed. Samples that were suspected to be Capripoxvirus positive were used and analyzed for this study. A total of 62 samples, comprising blood, skin scrapings, and cell culture isolates from cattle, sheep, and goats, were analyzed. The study compared the performance of a newly designed LAMP primer set (10_LSD), developed using the GLAPD software, with a generic Capripoxvirus (CaPV) primer and conventional molecular assays (PCR and qPCR, the gold standard). Appropriate positive and negative controls, which were a pure culture sample of lumpy skin disease virus and nuclease-free water, respectively, were included throughout all assays to ensure diagnostic accuracy and reliability. The 10_LSD LAMP primer demonstrated superior diagnostic performance with a sensitivity of 60% and specificity of 86%, compared to 56% and 50%, respectively, for the CaPV primer. The 10_LSD assay showed substantial agreement with qPCR, with a kappa value of 0.32, indicating fair agreement, while CaPV and PCR had kappa values of 0.04 (agreement equivalent to chance) and 0.17 (slight agreement), respectively. The 10_LSD assay also demonstrated a lower limit of detection (1 ng/µl) than the CaPV primer (10 ng/µl), though qPCR remained more sensitive (10⁻³ ng/µl). Time to positivity for the LAMP assay ranged between 14 and 28 minutes. The 10_LSD LAMP assay stands out as the best diagnostic option for Lumpy Skin Disease Virus (LSDV) due to its high specificity (86%), which minimizes false positives and ensures meaningful results. Its practicality for field use is a major advantage, requiring minimal equipment and providing results within 15–23 minutes. With a high positive predictive value (93.5%) and a strong negative predictive value (85.7%), it reliably guides treatment and control decisions. Additionally, a field-based DNA extraction method using polyethylene glycol (PEG) was developed for skin biopsies and paired with the LAMP assay. This study demonstrates that the 10_LSD LAMP assay is a promising diagnostic tool for rapid, on-site Capripoxvirus detection. The assay demonstrates proof of concept, validating its potential for further optimization and eventual field deployment. MSc in Biotechnology 2025-07-15T00:00:00Z