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Item Physico-Chemical and Microbiological Analysis of Honey and Tej Collected from Central Ethiopia and Production of Tej Using Starter Culture(Addis Ababa University, 2024-06) Frehiwot Dagima; Mogessie Ashenafi; Dagim Jirata; Asnake Desalegn; Fitsum TiguTej is the most diverse traditional fermented alcoholic beverage ofEthiopia. It ispreparedfrom honey, waterand leaves of Gesho (Rhamnusprinoides). In our country, Tej iscommonly fermented bynatural microflora. The aimof this study wasto investigate thephysicochemical,proximate and microbiological properties of honeyand Tejfrom centralpart of Ethiopiaandits surrounding and production ofTejbyusingstarterculture.pH. specific gravity,moisture content, ash, acidity,electrical conductivity, Hydroxyl methyl furfural, sugarsand alcoholcontents arethe main physicochemical andproximate parameters analyzed in honey and Tejsamples. Aerobicspore-forming bacteria, Aerobic Mesophilic bacteria, Enterbacteriaceae,Yeastand Mouldsand Total coliforms were themicroorganisms assessed fortheTejsamples.Atotal of 20 honey and 30 Tej samples were collected.The meanvalues ofpHandAcidityofTejandhoney rangedfrom3.45-4.20and1.0-2.7,JorTejandfrom2.96-4.45and17.87-52.15forhoney, respectively,Themeanmoistureandashcontentsof Tejandhoneyvariedfrom86.7-92.7and0-0.2,/01'Tej,80.01-84.75and0-0.3forhoneyrespectively. ThemeanvalueofECofTejrangedfrom0.44-0.76andthevaluesofhoneyrangedfrom0.27-0.92.TheHMFofhoney rangedfrom0.0-1.05,andthemeanofalcoholcontentrangedfrom8.62-14.49fortheTej samples.Yeastswerethedominantmicroorganismsfoundin theTejandtheywerenotdefectedinhoneysamples.The yeast countswere rangedfrom 5.60-8.00Cfu/g. Aerobic spore-forming bacteria (ASFB),TotalcoliformandEnterbacteriaceaewere not detected Basedon theirgrowthperformanceunderdifferent physiologicalstressestenLAB and ten yeast isolateswerescreened. The Yeast isolates were tentativelyidentified intofour species level, Saccharomyces cerevisie, Saccharomyces daireness,Debaryomycescarsonii. BoththeLAB andyeast isolates were combined in differentproportions toformulate starter culturesforthe production of Tej. Tenformulations were made in different proportions based ontheir compatibility of theisolates. Using Four Yeast and Ten LAB-Yeast starter cultureformulations, Tej was prepared under controlled fermentation conditions. The overallsensoryacceptability analysisshowedthatformulate.F#5,F#2,andF#7werethebestmixedstarter cultures forTejpreparation as comparedwiththe control.However, further molecular identification of theisolatesintospecies level and investigation ofthemicrobialdynamics ofTejisrecommendedforfutureuseofthese isolates, The result forphysicochemical and proximateanalysiswere alignedwith nationaland internationalstandards.Item Characterization of Morphology, Genomic Diversity and Environmental Adaptation of Indigenous Cattle Populations in Tigray National Regional State, Ethiopia(Addis Ababa University, 2024-01) Tsadkan Zegeye; Gurja Belay; Olivier HanotteEthiopia is the home of the largest cattle population in Africa and the fifth in the world. Cattle in Ethiopia are the primary agricultural entity serving fully or partially the livelihood of around 70% of the population. They are the most important generators of the agricultural GDP, contributing around 80% of the annual production of milk and meat. However, despite providing the majority of the livestock products in the country, Ethiopia's indigenous cattle genetic resource has yet to receive much attention. Breed identification and characterization still need to be completed, and an institutional framework for conservation and wise management of their genetic diversity needs to be established. The Tigray National Regional State, in the North of Ethiopia, is the fourth cattle-populated region, with about 8% of Ethiopia's cattle genetic resources. However, Tigray is one of the regions of Ethiopia where its animal genetic resources still need to be fully identified and characterized. This study aimed to undertake a comprehensive characterization of indigenous cattle populations in Tigray (Abergelle, Arado, Begait, Erob and Raya) involving their morphology, ecological niche suitability, genome-wide genetic diversity, and the genomic response of selection to the environmental challenges. Sampling sites were selected purposively to include a comprehensive representation of the indigenous cattle populations from their respective natural breeding areas. A total of 1650 matured cattle from the five populations were included to investigate the phenotypic description based on qualitative and quantitative traits. Data analysis was performed using chi-square involving crosstabs for the qualitative variability and multivariate discriminant analysis involving GLM, STEPDISC, CANDISC, DISCRIM, and Canonical discriminate function procedures of SAS for the quantitative variability. The stepwise discriminant analysis screened eighteen variables with a discriminant power for characterizing the female and thirteen for the male populations. High correct assignments to source populations were obtained for all populations except Abergelle and Erob, where around 30% of each shared morphologic similarity. The five populations were also clustered into four populations, with Abergele and Erob cattle overlapping. The environmental niche of each cattle population was characterized by the new approach applied to livestock, Environmental Niche Modeling (ENM). From the sampling locations, thirty coordinates (4 to 7 per population) were collected using the Geographic Positioning System (GPS) and nine coordinates surrounding 1 kilometer of the initial sampling location were extracted using Google Earth Pro 7.3.1.4507. Finally, 300 coordinates were used to extract data (from thirty-three environmental predictors) for habitat suitability mapping and screening out of the main environmental variables. Four distinct habitat suitability maps were detected, except for the Arado and Erob cattle, with around 66% niche similarity. Six main environmental variables, temperature seasonality, soil bulk density, cultivated land, and annual, wettest and warmest quarter precipitations that could have a potential driving factor for morphological and genetic variability across the indigenous cattle in Tigray were sorted out. Next, the whole genome sequence data was followed to characterize the genome-wide genetic diversity, relatedness, and admixture of the same five cattle populations. A high number of genetic variants were detected, where around seven and thirty-four per cent of SNPs and indels were novel, respectively. The genome-wide average nucleotide diversity ranged from 0.0035 to 0.0036. The number of heterozygous SNPs was about 0.6 to 0.7 higher than homozygous SNPs. There was high variability in ROH records among and within animals of each population, with the lowest record in Arado (777.82) and the highest in Raya (1000.45). Similarly, the analysis of inbreeding revealed differences within and among populations, ranging up to 10% in some individuals of Begait and Raya populations. Only a fraction (0.01% SNPs and 0.22% to 0.27% indels) of the identified variants annotated for functional variability overlapped coding regions. The enrichment analysis of genes overlapped missense private SNPs screened 20 significant GO terms and KEGG pathways common or specific to each population. Out of the gens overlapped missense private SNPs, the genes SCN4A, TAS1R2 and KCNG4 related to body size and length were specifically detected in Begait cattle and genes MMRN2 and VWC2 related to meat quality were detected in Erob cattle supporting the morphological finding. The population structure revealed the ancestry background of the indigenous cattle in Tigray from Asian indicine (85.6% to 88.7%) and African taurine (11.3% to 14.1%) cattle, with very small European taurine introgression in some individuals. Finally, the positive signatures of adaptation of the cattle populations to the main environmental stressors were analyzed following two genomic scans (Hp) and (FST). Selective sweeps of the overlapped regions were analyzed using Hp and FST to retrieve the candidate protein-coding genes. Around 60% of the annotated selective sweeps regions overlapped with protein-coding genes, while the rest lacked genes. GO and KEGG pathways of the protein-coding genes overlapped selective sweep regions revealed enriched (P < 0.05) genes involved in adaptation to moisture-stressed lowlands (HELB, HMGA2, IRAK3, LLPH, UCN2, LOC101902172, ADAMTS16, DDB1, ASIP, IL17B, SNAP29) and moisture-stressed highlands (NQO1, NEK6, LHX2, UCP2, UCP3 and LCMT2). This study shared detailed findings on the morphology, genetic and adaptive diversity of the indigenous cattle in Tigray. Diverse morphological and genomic diversity was observed in the Indigenous cattle in Tigray, indicating their importance as a genetic reservoir at regional and country levels. Moreover, the production type of the indigenous cattle in Tigray screened through the interlinking of morphological description and specific genes selected for production values provide insight into their breeding management.Item Therapeutic Efficacy of Artemether-Lumefantrine for the Treatment of Uncomplicated Plasmodium Falciparum Malaria in Metehara Town, Central-East Ethiopia(Addis Ababa University, 2024-03) Mahelet Tesfaye; Hassen Mamo; Ashenafi AssefaMonitoring and identification of drug-resistant Plasmodium falciparum strains is paramount for the fight against malaria. Close surveillance of the emergence and distribution of artemisinin resistance is recommended to guide policy decisions. The efficacy of national first- and second-line anti-malarial treatments should be monitored at least once every 2 years, as recommended in the WHO standard protocol. In Ethiopia, a three-day regime of AL (artemether 20mg and lumefantrine 120mg in each tablet) is the first-line anti-malarial drug for the treatment of uncomplicated P. falciparum malaria since 2004. The objective of this study was assessing the therapeutic efficacy of AL for the treatment of uncomplicated P. falciparum malaria in Metehara, central-east Ethiopia. The study was conducted at Metehara town health center from November 26, 2020 to March 24, 2021. One-arm prospective evaluation was conducted on the clinical and parasitological responses to directly observed treatment for uncomplicated P. falciparum malaria. During the study regime, 80 patients were screened and 73(50 male and 30 female) participants completed the follow-up and among those 14 patients were <5 age, 25 between the age 5-14 and 34 were >14. The overall cure rate was 100% (73/73; 95% CI: 95.1-100.0) with no early treatment failure, late treatment failure, and late parasitological failure as in Kaplan–Meier analyses all participants completely recovered from parasitemia and fever on day (D) 3; the asexual parasite clearance rate was 100% and clinical symptoms resolved quickly. Gametocyte carriage was reduced from 8.4% on D0 to 1% on D3 and complete clearance was achieved on D7. There was no serious adverse event. In the study location, AL was effective for treating uncomplicated P. falciparum malaria.Item Hodgkin Lymphoma at a Tertiary Cancer Center in Ethiopia: Novel Biomarkers and Characterization of the Tumor Microenvironment in Relation to Epstein Barr Virus (EBV)(Addis Ababa University, 2024-05) Makka Adam; Beyene Petros; Rawleigh Howe; Mats Jerkeman; Amha G/Medhin; Yonas BekuretsionClassificahon and idenhficahon of Hodgkin lymphoma (HL) relies on morphological and biomarker-based diagnosis. However, CD30 and CD15 used for the idenhficahon of HL, are not expressed on all tumor cells. This gap can be narrowed by combining detechon of the insulin-like growth factor II messenger RNA-binding protein 3 (IMP3). This study aimed to confirm the diagnoshc value of IMP3. In addition to investigating the prevalence of EBV among HL cases, the tissue cellular composition of EBV-related and EBV-unrelated cases also was assessed. Furthermore, the treatment outcomes and prognostic impact of FoxP3 and PD1 in the tumor microenvironment (TME) of classical HL (CHL) were determined. Clinical records of consecutive patients with HL diagnosed between the years 2014 and 2019 were reviewed. A tissue microarray (TMA) of 126 of CHL and nodular lymphocyte predominant HL from Tikur Anbessa Hospital was constructed. TMA sections were usedfor immunohistochemical staining and detection of Epstein Barr Virus encoded-RNA. The stained immune cells were quantified by HALO 2.3. A total of 91(68.4%) pahents were male and the male-to-female raho was 2.2:1 with a median age of 22 years. The majority of the cases, 67 (50%) were of the mixedcellularity and 40 (40%) of the nodular-sclerosis subtype. A total of 77 (61.1%) of HL cases expressed LMP1/EBER. The immunoreachvity of HL cases to IMP3 was determined and compared with the commonly used biomarkers for HL diagnosis. 122 (96.8%), 95 (75.4%), and 126 (100%) of the cases were posihve for CD30, CD15, and IMP3 markers, respechvely. Infiltration of CD8+, T-bet+, and FoxP3+ cells was higher in the TME of EBV-related CHL, with P values of <0.001, <0.001 and <0.016, respectively. In contrast, PD1 expression was higher in the TME of EBV-unrelated CHL (P < 0.001). In a Kaplan-Meir analysis, the 9-year overall survival (OS) and event free survival (EFS) was 78.6% and 66.5%, respectively. The patients in the high-risk group, International Prognostic Score (IPS ≥ 3), had inferior OS and EFS compared to the patients in the low-risk group (IPS< 3) (P= 0.04 for both survival outcomes). HIVassociated HL had inferior EFS (P= 0.016). Patients with low lymphocyte (≤ .6x109 cells/L) had inferior OS (HR= 10.9; 95% CI, 1.4-84; P= 0.016) and EFS (HR= 5.9; 95% CI, 1.7-20; P= 0.005). Patients with high FoxP3+ cells (≥ 9%) and high PD1+ cells (≥ 24.6%) in the TME of CHL had poor prognosis. The 9-year OS for high FoxP3 was 64.6% with (HR= 4.3; 95% CI, 1.2-15.7; P= 0.02) and the 9-year OS for high PD1 was 57.1% with (HR=3.4; 95% CI, 1.2-15.7; P=0.03). Low lymphocyte count, high FoxP3+, and high PD1+ were all significantly associated with adverse overall survival in a multivariate Cox-regression. The present study supports previous reports that suggest the potenhal of using IMP3 as a diagnoshc biomarker for HL. The study findings further highlight the previously unrecognized possibility that distinct immunosuppressive mechanisms involving FoxP3+ and PDI-expressing cells may be in play within EBV-positive and negative HL types. Although the treatment outcomes is relahvely inferior compared to high-income countries, a significant proporhon of pahents with CHL, are cured when provided adequate treatment. Overall, HIV-associated CHL, low lymphocyte count, high FoxP3, and PD1 were associated with unfavorable treatment outcomes.Item Mycobacterium Tuberculosis Infection Among Homeless Individuals in Addis Ababa, Ethiopia: Disease Burden, Drug Resistance Patterns and Molecular Epidemiology(Addis Ababa University, 2024-05) Tsegaye Shamebo; Beyene Petros; Gobena Ameni; Balako GumiIn high tuberculosis (TB) burden countries like Ethiopia, rapid screening and prompt treatment initiation among vulnerable groups, such as the homeless, are essential for TB control efforts. During the last three decades, Ethiopia has experienced a rise in homelessness, which is attributed to internal conflicts and economic stress. In spite of the fact that TB disproportionately affects homeless individuals, the majority of research conducted on it in Ethiopia has not adequately addressed the disease burden on this vulnerable group. This study aimed to determine the disease burden, molecular epidemiology, and drug resistance patterns of Mycobacterium tuberculosis (M. tuberculosis) among homeless individuals in Addis Ababa, Ethiopia. A cross-sectional study was conducted in Addis Ababa between February 2019 and December 2020. Homeless individuals underwent pulmonary tuberculosis (PTB) clinical screening according to WHO guidelines. Suspected cases provided sputum samples for acid-fast bacillus (AFB), Xpert MTB/RIF assay, TB culture, and drug sensitivity test (DST). The M. tuberculosis isolates were typed using Polymerase-Chain-Reaction (PCR) based Region of Difference-9 (RD9), spoligotyping, and 24-loci M. tuberculosis Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing. DST was performed using the BD Bactec Mycobacterial Growth Indicator Tube (MGIT) 960. Data analyses were performed using SPSS software version 26 and the M. tuberculosis complex (MTBC) online database. Out of 5,600 homeless individuals enrolled in the study and clinically screened for PTB symptoms, 641 suspected cases were identified. Thus, the clinical prevalence of PTB was 1054 per 100,000 homeless individuals. Being homeless for more than 5 years, a body mass index (BMI) < 18.5, smoking cigarettes, living in a group of more than 5 persons, close contact with chronic coughers, imprisonment, and HIV infections were significantly associated with the prevalence of PTB in the homeless (P < 0.05). Out of 59 isolates, 58 were confirmed as M. tuberculosis by the RD9 PCR test. Genotyping revealed three MTBC lineages and eight sub-lineages, with Euro-American lineage predominating. Furthermore, Spoligo International Types (SIT), SIT53, SIT37, and SIT149 were highly prevalent strains detected in this study. Ethiopia_3, Delhi/CAS and Ethiopia_2 were determined to be the most prevalent sub-lineages in the study population. Strain clustering rates were 77.6% using spoligotyping, 39.7% using 24-loci MIRU-VNTR typing, and 10.3% using a combination approach. Living in a group was significantly associated with strain clustering (P < 0.05). Three homeless individuals with PTB harbored mixed M. tuberculosis strains. DST revealed 6.8% (4/59) of isolates resistant to at least one first-line anti-TB drug. Overall, the prevalence of PTB in homeless individuals was higher than that in the general population of Addis Ababa. Therefore, governmental and non-governmental organizations working on TB prevention and control must consider homeless settings as hotspots for TB control. Regular PTB screening, directly observed treatment short course (DOTS) centers, and mobile clinics must be established to control TB among homeless individuals and its spread to the general population.Item Breast Cancer Subtypes, Associated Biomarkers, and the Involvement of Human Papilloma Virus in Ethiopian Population(Addis Ababa University, 2023-12-22) Esmael Besufikad; Adey Feleke; Tesfaye Sisay; Rawleigh Howe; Dinkisira BekeleBreast cancer is the most common type of cancer in the world as well as in Ethiopia. Although research on breast cancer in Ethiopia has been conducted, none of them have evaluated breast cancer in multiple regions of the country, which is important considering Ethiopia’s enormous ethnic and genetic diversity. Hence, this study was carried out to evaluate the distribution of breast cancer subtypes and associated immune cell biomarkers, hormone receptors, matrix metalloproteinases (MMPs), and HPV genotypes in selected Ethiopian regions. A total of 227, 81, 58, and 120 formalin-fixed paraffin-embedded (FFPE) tissue blocks were collected for breast cancer subtyping, immune cell biomarkers analysis, MMP expression, and HPV genotyping, respectively. Immunohistochemistry (IHC) staining was performed for breast cancer subtyping based on estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), and Ki-67 proliferation markers, and for additional immune cell biomarker expression. RNA was extracted and quantitative reverse-transcription PCR was performed for MMP expression analysis. DNA was extracted from archived FFPE breast tissue specimens and target genes were amplified using PCR for HPV genotyping. SPSS Version 25 was used to enter and analyze data. For immune cell biomarkers and MMP results, GraphPad Prism version 8.0.0 was used for statistical analysis. A large percentage of breast cancers were found to have advanced clinical and pathologic features, such as substantial lymph node involvement, large tumor size, and high histological grade. The percentage of ER and PR-negative tumors were 48.3% and 53.2%, respectively. The IHC subtype distribution was 33.1% triple-negative (ER-, PR-, HER-2-) breast cancer, 27.6% luminal B ((ER+, PR+, HER-2- and Ki-67 ≥ 20%) or (ER+, PR+, and HER-2+)), 25.2% luminal A (ER+, PR+, HER-2- and Ki-67< 20%), and 14.1% HER-2-enriched (ER-, PR-, HER-2+). In multiple logistic regression analysis, grade III and HER-2 positivity were associated with larger tumor size, and tumor size was also higher in samples from Southwestern Ethiopia (Jimma) as compared to Northern Ethiopia (Mekele). The MMP-11 expression levels were significantly higher in breast cancer cases than in benign breast tumors (P=0.012). The non-luminal (triple-negative and HER-2-enriched) breast cancer subtype had a higher percentage of stromal CD20+, intratumoral CD3+ tumor-infiltrating lymphocytes, and CD68+ tumor-associated macrophages than the luminal (Luminal A and Luminal B) subtype. The stromal programmed cell death ligand 1 (PD-L1) +, intratumoral CD3+ tumor-infiltrating lymphocytes, CD163+ tumor-associated macrophages, and PD-L1+ were also more commonly found in grade III breast cancer than in grade I and II breast cancer, respectively. Human papillomavirus was found in 20.6% of breast cancer patients and 29.6% of non-malignant breast tumors. Human papillomavirus infection was nearly 10-fold more common in ER-positive than ER-negative breast cancer. A considerably high prevalence of triple-negative breast cancer was reported in our study, demanding additional research that includes identifying genetic predisposition factors. A significant association was found between the breast cancer subtype and stromal CD20+, intratumoral CD3+ tumor-infiltrating lymphocytes, and CD68+ tumor-associated macrophages. The stromal PD-L1+, intratumoral CD3+ tumor-infiltrating lymphocytes, CD163+ tumor-associated macrophages, and PD-L1+ were also associated with tumor grade. Our findings suggest an important impact of MMPs in breast cancer pathophysiology, particularly MMP-11. This study also showed no proof of a link between HPV infection and breast cancer; however, the finding that HPV was more prevalent in breast tumors that were ER-positive than ER-negative warrants further attention.Item Bioactivity of Essential Oils of Thymus Serrulatus and Thymus Schimperi from Ethiopia: Hepatoprotective, Dental Caries Protective, Mosquitocidal and Acute Oral Toxicity(Addis Ababa University, 2016-01) Destaw Damtie; Yalemtsehay MekonnenThis study was conducted to investigate; the ethnobotanical information, chemical composition, bioassays (antibacterial, mosquitocidal, larvicidal, and oviposition deterrent, hepatoprotective) and acute oral toxicity of the essential oils (EOs) of Thymus species collected from six localities in Ethiopia. Ethnobotanical information was collected using semistructured questionnaires, antibacterial test using disk diffusion technique, mosquitocidal activity using fumigation test, hepatoprotective activity in male Wistar rats and acute oral toxicity in female albino mice. Thymus species collected from Ofla (Ofl), Alamata (Ala), and Yilmana Densa (Yil) were identified as Thymus serrulatus and those collected from Tarmaber (Tar), Butajira (Buta), and Bale (Bal) as Thymus schimperi. Both species which are endemic to Ethiopia are traditionally used to treat different illnesses like blood pressure, general pain syndrome, liver diseases, influenza, abdominal pain, and against intestinal parasites. The major compounds in Ofl EO were thymol (49.55%), carvacrol (36.34%), and p-cymene (3.06%). In Ala EO, thymol was the dominant component (65.63%) followed by carvacrol (6.68%) and thymol methyl ether (6.55%). Yil EO on the other hand had carvacrol (80.84%), thymol (6.52%), and p-cymene (3.65) as its major components. Tar was the EO with thymol (48.84%), carvacrol (42.12%), and linalool (2.97%) as its major components. In the same way, the major components of Buta EO were (71.83%), thymol (15.77%), and p-cymene (3.75%). The predominant components of the last EO, Bal were thymol (53.57%), carvacrol (34.55%), and p-cymene (3.20%). Four of the EOs (Ofl, Ala, Tar, and Bal) were found to be thymol and the rest two (Yil and Buta) carvacrol chemotypes. All the essential oils inhibited cariogenic bacteria (Streptococcus mutans and Lactobacillus). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the Bal EO against S. mutans was found to be 0.25 μL/mL and the MIC/MBC of all the rest EOs against this bacterium was 0.5 μL/mL. On the other hand, the MIC and MBC of all the EOs against Lactobacillus was at the dose of 0.5 μL/mL. Paracetamol-induced hepatotoxicity in Wistar rats was prevented by the application of EOs at 200 μL/Kg body weight. This was demonstrated by the reduced serum levels of marker enzymes; alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). In addition, it was seen from the histopathological tests that Thymus EOs prevented paracetamol-induced necrosis. Thymus EOs too showed larvicidal, mosquitocidal (fumigation test) and oviposition detterent activities against the mosquito Anophelus arabiensis. The essential oil concentrations that resulted in mortality of 50% of larvae (LD50) were: (60.75 μL/L, Ala); (44 μL/L, Yil); and (41.75 μL/L, Tar). The LD50 values for the fumigation test, on the other hand, were: (17.19 μL/L, Ala); (14.92 μL/L, Yil); and (13.20 μL/L, Tar). The 200 μL/L, 100 μL/L, and 50 μL/L doses of Tar; the 200 μL/L and 100 μL/L doses of Ala; and the 200 μL/L dose of Yil resulted in complete oviposition detterent activity. Except some irritation responses, all the test EOs were found to be non-toxic to mice and had LD50 values in the range of 2000μL/kg body weight to 5000μL/kg body weight with corrected acute toxicity point (LD50) estimate of 2500μL/kg body weight. In conclusion, the different chemotypes of T. serrulatus and T. shimperi EOs resulted in antibacterial activities, hepatoprotective activities, mosquitocidal activities and were not toxic.Item Culturable Endophytes of Enset (Ensete Ventricosum): Diversity, Plant Growth Promotion, and Bacterial Wilt Disease Control Potentials(Addis Ababa University, 2015-02-05) Yemisirach Mulugeta; Amare Gessesse; Appollinaire DjikengEnset (Ensete ventricosum (Welw) Cheesman) is a perennial root crop used as a staple and co-staple food in Ethiopia. Enset cultivation is one of the sustainable agricultural systems in the country and offers significant ecological benefits by reducing soil erosion, resistance to different diseases and pests and relative tolerance to drought. Although enset agriculture is a good example of sustainable agriculture, the crop is largely affected by bacterial wilt disease caused by Xanthomonas campestris pv. musacerum. Because the pathogen resides in the vascular tissue of host plant, chemical based control strategies cannot be employed to control the disease. Studies showed that endophytes, symbiotic microorganisms (both bacteria and fungi) that live inside healthy plants have properties of biological control against different plant diseases. Having these properties endophytes might be one component of integrated disease management programs on enset. This study was initiated with the aim of investigating the diversity of culturable endophytic bacteria and fungi from enset and assesses the presence of phytobeneficial properties and their roles in protecting the plant against enset wilt disease. Endophytic bacteria and fungi were isolated from surface sterilized samples of leaf, stem and root of healthy enset plants. A total of 446 bacterial and 105 fungal endophytes were identified indicating the presence of high diversity of bacterial and fungal endophytes associated with enset. The bacterial endophytes were grouped to four phyla and 53 genera. Proteobacteria were the most frequently isolated phylum from all the plant parts followed by gram positive Actinobacteria, Firmicutes, and non-proteobacteria gram negative Bacteroidetes. The fungal isolates were grouped in to two phyla and 42 species. Ascomycota was more detected phylum compared to Zygomycota. The in vitro investigation of plant growth promotion (PGP) characteristics of 105 bacterial and 44 fungal isolates showed that majority of the isolates were positive to one or more plant growth promoting characteristics. IAA production is a more common property among enset endopytes. Moreover, there were isolates that produce siderophore and able to grow on nitrogen free medium. Phosphate solublisation was detected in bacterial isolates but none of the tested fungal endophytes show phosphate solublization. The potential of enset endophytes to control bacterial wilt of enset was also assessed in vitro and in vivo. In the in vitro test bacterial and fungal endophytic isolates belonging to different genera showed marked inhibition of the growth of Xanthomonas campestris pv. musacerum with the highest observed for bacterial isolates belonging to the genera Pseudomonas, Bacillus, and Rhizobium. And from the fungal endophytes two isolates identified as Mycosphaerella coacervata and Plectosphaerella cucumerina inhibited the growth of the pathogen. Plants treated with a mixed culture of bacteria showed a mean disease severity of 30.7% as compared to a disease severity of 47.4% for the control plants. In the endophytic metabolite analysis for potential antimicrobial compounds, hundreds of VOC and NVDOC were identified. The compounds produced include phenols, lactone, cyanide, pyrazine and dimethyl disulfide. Some of the compounds were reported to have antimicrobial activity against different fungal and bacterial pathogens in previous studies. Moreover, other unknown compounds were also detected In conclusion, this study showed high occurrence, diversity, plant growth promoting characteristics and the biocontrol potential of enset endophytes. Furthermore, the results also indicated enset endophytes as a potential alternative increase crop productivity.Item Molecular Epidemiology and Drug Resistance Analysis of Mycobacterium Tuberculosis Isolates from Central, Eastern and Southeastern Ethiopia(Addis Ababa University, 2024-05) Mulualem Agonafir; Gurja BelyUnderstanding the prevalent lineages, drug resistance mutations, and transmission dynamics of Mycobacterium tuberculosis complex (MTBC) across diverse regions is crucial for designing effective tuberculosis (TB) control strategies. In Ethiopia, comprehensive studies on the molecular epidemiology and drug resistance of TB are scarce, with existing research focused on specific regions like Addis Ababa and the north/southwestern parts. This study aimed to assess genetic diversity, transmission patterns, and drug resistance mutations among Mycobacterium tuberculosis complex (MTBC) isolates in central, eastern, and southeastern Ethiopia. Conducted between August 2018 and January 2019, it involved 232 culture-positive MTBC isolates from pulmonary TB patients referred to Adama and Harar TB reference laboratories. Spoligotyping identified prevalent lineages and sub-lineages, revealing a diverse population structure with six major lineages. The Euro-American (Lineage 4) and East-African-Indian (Lineage 3) lineages were dominant, comprising nearly 95% of isolates. High genetic diversity was observed, with 77 distinct spoligotype patterns. Dominant spoligotypes included SIT149/T3_ETH, SIT53/T1, SIT21/CAS1_Kili, and SIT41/Turkey, with a rare Beijing spoligotype (SIT541) detected in eastern Ethiopia. Strain clustering was significantly associated with individuals aged 25-34 years. Genotypic drug susceptibility testing identified mutations conferring resistance to rifampicin (RIF) and isoniazid (INH) in nearly 40% of isolates. Mutations for resistance to fluoroquinolones (FLQs) and second-line injectable drugs (SLIDs) were less frequent, observed in around 9% and 4% of isolates, respectively. Dominant mutations included S531L in rpoB for RIF, S315T in katG for INH, A90V in gyrA for FLQs, and WT1 in rrs for SLIDs. WGS of multidrug-resistant (MDR) MTBC isolates supported these findings, revealing detailed information on specific drug resistance mutations for an expanded list of drugs. The Euro-American (Lineage 4) and East-African-Indian (Lineage 3) remained the most prevalent genotypes among MDR MTBC isolates in East Ethiopia. Core genome Multi-locus Sequence Typing (cgMLST) analysis revealed recent MDR-TB transmission events in 46.7% of clustered isolates, with a high proportion from Diredawa city, suggesting localized transmission. One cluster was the Beijing sub-lineage of East Asian (Lineage 2), known for high transmission and drug resistance. In conclusion, this study highlights the diverse genetic structure and significant drug resistance mutations of MTBC isolates in central, eastern, and southeastern Ethiopia. The findings emphasize the dominance of specific lineages and sub-lineages, the presence of diverse mutations, and localized transmission hotspots. This underscores the need for tailored control strategies and comprehensive molecular surveillance to effectively address TB and MDR-TB in Ethiopia.Item Evaluation of Beauveria Bassiana Isolates Against Varroa Destructor of Apis Mellifera Under Laboratory Condition(Addis Ababa University, 2024-04) Olyad Daniel; Asnake DesalegnVarroa, Varroa destructor (Acari: Varroidae), is an ectoparasitic mite of honeybees, Apis mellifera. This parasite poses a substantial threat to the health, welfare and production of A. mellifera globally, including Ethiopia. Without chemical treatment, colony losses worldwide are common. However, resistance to synthetic chemicals in beekeeping is increasingly concerning. Entomopathogenic organisms provide an eco-friendly alternative to pesticides and can prevent resistance development. Therefore, isolates of Beauveria bassiana were tested for their pathogenicity against V. destructor and their negative effect on the brood and adult stages of the central highland honeybees of Ethiopia, A. mellifera. Varroa mites were immersed in 5 millilitres of a conidial suspension containing 1 x 108 conidia/mL of three different fungal isolates (APPRC-44BC, APPRC-27, and S#10H), as well as control solutions (0.05% Tween 80 and distilled water). The inoculated mites were placed on honeybee brood inside capped cells. Then, the infected brood combs were kept in an incubator at 33 ℃ and 60% relative humidity for ten days. The fungal isolates and control treatments were also applied to young adult workers and healthy brood to observe the effects of the treatments. Fungal isolate APPRC-44BC displayed the highest (73%) potential for killing varroa mite. All three isolates (APPRC-44BC, APPRC-27, and S#10H) were found to be highly efficient between the sixth and eighth days post-application, accounting for 96.8% to 100% of the fungal-induced deaths. Interestingly, treatment with B. bassiana isolates did not show a significant effect on brood emergence and the weight of newly merged adults. However, the treatments had significant effect on adult honeybee survival. It is clearly observed from these results that isolates of B. bassiana are potential bio-control agents against V. destructor. However, further studies are needed to evaluate the efficacy of this promising fungal isolate (APPRC-44BC) in honeybee colonies under field conditions, as well as to develop application methods that have minimal impact on adult honeybee survival.Item Zoonotic Cryptosporidium Species Infections in Humans and Sympatric Non-Human Primates, Chlorocebus Aethiops and Colobus Guereza in Wurgissa and Hawassa, Ethiopia(Addis Ababa University, 2023-06) Ambachew Woreta; Beyene PetrosCryptosporidiosis has become a significant public and veterinary health concern worldwide. In Ethiopia, non-Human Primates (NHPs), Chlorocebus aethiops and Colobus guereza monkeys are now living in overlapping/adjacent geographical areas to human settlements and no studies in Ethiopia have assessed Cryptosporidium infection at species or genotypes level on NHPs. Therefore, this cross-sectional study was conducted to determine the prevalence of Cryptosporidium spp. infections, identify species and subtypes in both NHPs and humans in Ethiopia. Fecal samples were collected from 187 humans (94 from Wurgissa and 93 from Hawassa) and 185 NHPS (147 from Wurgissa and 38 from Hawassa) in Ethiopia. Humans who visited health centers (WHC and HHC) and volunteered to participate were interviewed and screened for the presence of Cryptosporidium spp. using modified Ziehl–Neelsen technique. All samples were submitted to Rouen University Hospital, France, for molecular analysis using DNA sequencing of the SSU 18S rRNA and Gp60 genes. The overall prevalence of infection was 46% (n = 86) by microscope and PCR. When 48 (out of 86) PCR positive samples were genotyped, two species were identified: C. parvum (n = 40) and C. hominis (n = 8). When 15 of the 40 C. parvum isolates were subtyped, zoonotic subtypes of IIaA14G1R1 (n = 1), IIaA15G2R1 (n = 1), IIaA16G1R1 (n = 2), IIaA16G3R1 (n = 2), IIaA17G1R1 (n = 1), IIaA19G1R1 (n = 1), IIaA20G1R1 (n = 3), IIaA22G1R1 (n = 1), IIaA22G2R1 (n = 1), IIdA23G1 (n = 1) and IIdA24G1 (n = 1) were identified. When 6 of the 8 C. hominis isolates were subtyped, subtypes IaA20 (n = 5), and IdA21 (n = 1) were identified. The overall prevalence of Cryptosporidium spp. infection in NHPs using molecular detection was 70.9 % (131/185). Of the 131 Cryptosporidium spp. infection in NHPs, 54 have been characterized and sequence analysis showed C. parvum (n=36), C. hominis (n=15), and C. cuniculus (n=3). C. parvum IIa subtype family (IIaA17G1R1) was the common subtype in NHPs. Also, multiple subtype families of C. hominis (Ia, Ib, Id, and Ie) were documented. Humans who had contact with NHPs and diarrheal person, family size and education status were major risk factors associated with Cryptosporidium spp. infection. The present study has provided strong evidence that NHPs in Ethiopia harbor Cryptosporidium spp. of public health importance and can be considered potential reservoirs of human cryptosporidiosis. Furthermore, the predominant occurrence of C. parvum in humans and NHPs and the existence of C. hominis are suggestive of zoonotic transmission. Thus, studies involving experimental cross infections of parasite isolates from both hosts and molecular characterization of the parasites can provide definitive evidence for the role of NHPs in the transmission of Cryptosporidium spp. to humansItem In Vitro Antipromastigote Activity of Methanol Extracts of Malva Parviflora Leaves and Roots Against Leishmania Donovani and Leishmania Aethiopica(Addis Ababa University, 2024-01) Yeabtsega Tilahun; Gurja Belay; Tegenu Gelana; Solomon Mequanente; Yehenew Asmamaw (PhD)Leishmaniasis is a neglected tropical disease threatening the lives of about 350 million people, globally. There is a small selection of medications on the market that can be used to treat leishmaniasis. However, the currently available drugs are limited in number and have drawbacks including variable efficacy, toxicity and unaffordability which call upon search for viable options. The leaf and root of Malva parviflora are used for the treatment of leishmaniasis in traditional medicine in Ethiopia. The aim of this study was to evaluate the antipromastigote activities of methanol extracts of Malva parviflora against Leishmania promastigotes. Fresh leaves and roots of Malva parviflora were chopped and then macerated with sufficient amount of 100% methanol at room temperature. Clinical isolates of L. donovani and L. aethiopica were obtained and the isolates were grown in tissue culture flasks containing RPMI 1640 medium supplemented with fetal bovine serum. L. aethiopica and L. donovani at the logarithmic stages were inoculated in to liquid media for the assay. The percent inhibition of the growth of promastigotes of Leishmania species of the methanol extracts of leaf and root of M. Parviflora evaluated in vitro, at concentrations of 100 μg/ml and investigated for their anti-leishmanial activities against promastigotes of L. aethiopica and L. donovani and for their cytotoxicity. The percent of inhibition of leaf of Malva parviflora was 55.89 ± 2.19% and 79.84 ± 1.57% and the root of M. parviflora was 74.21 ± 2.39% and 78.81 ±1.88% respectively. The leaf extract has an IC50 of 1.73 and an IC50 of 0.002 against both promastigotes of L. aethiopica and L. donovani respectively. The IC50 of the root extract against L. aethiopica and L. donovani are 0.05 and 0.002 respectively. The leaf extract had a hemolysis percentage of 21.05 ± 11.54% and the root extract had 0 ± 26.54% percentage hemolysis of the extracts against human red blood cells. The leaf extracts had a selective index of 75.2 against L. aethiopica and a selective index of 260,200 against L. donovani isolates. The root extracts exhibited a selectivity index of >1000 against both L. aethiopica and L. donovani. The findings of this study conclude that the root extracts of Malva parviflora exhibit higher anti-leishmanial activities against both Leishmania strains and was found to be less toxic against human red blood cells when compared with the leaf extract. Further investigations on the bioactive compounds and fractions of M. Parviflora are recommended.Item Epidemiological and Molecular Characterization of Multi-Drug Resistant Gram-Negative Bacterial Isolates from Bloodstream Infections Among Patients Admitted at Tikur Anbessa Specialized Hospital, Addis Ababa, Ethiopia(Addis Ababa University, 2024-03) Daniel Beshah; Adey Desta; Tesfaye Sisay; Gurja BelayBloodstream infections are the major causes of morbidity and mortality worldwide. Alarmingly, Gram-negative bacteria that produce beta-lactamase and carbapenemase are causes for the rapid global spread of multi-drug resistance (MDR), which significantly jeopardizes the efficacy of existing antimicrobial treatments. This research aimed to analyze the microbial profile, antimicrobial susceptibility, genomic diversity, and associated risk factors of Gram-negative bacteria in bloodstream infections. A cross-sectional study was conducted between September 2018 and March 2019 on 1486 bloodstream infection suspected patients. In addition to biochemical identification and antimicrobial susceptibility tests, PCR and WGS were conducted for ESBL, AmpC, MBL and carbapenemase producing resistance genes. The prevalence of bloodstream infection was 417 (28.06%), and the most prevalent bacterial species were Klebsiella pneumoniae (17.6%) and Acinetobacter spp. (11%). Culture positivity was associated with age below 6 years, ICU admission, length of admission > 5 days, temperature greater than 38 oC, instrument usage during medical care, chronic illness, and neonatal incubation. Multi-drug resistance was 95% where 56%, 32% and 7% of the isolates exhibited MDR, XDR, and PDR respectively. Klebsiella pneumoniae and Acinetobacter sp. showed the greatest rates of XDR (37%) and PDR (77%), respectively. Among the 231 phenotypically characterized isolates, 195 (84%) drug-hydrolyzing enzyme producers, 54% and 26% were ESBL and carbapenemase-producer, respectively. Again, Klebsiella pneumoniae was the highest drug-hydrolyzing enzyme-producer bacteria. 176 out of the 195 (76%) were PCR-confirmed resistant bacteria, of which ESBLs, MBLs, carbapenemase, and AmpC-BLs accounted for 53%, 22%, 20%, and 10%, respectively and blaCTX-M, blaFOXM, blaOXA-23, and blaNDM were the highest hits in Klebsiella pneumoniae, (75%), and Escherichia coli (24%). Out of the 142 whole genome sequenced isolates, 1604 genes categorized into 168 resistance classed were identified. A total of 1055 virulence genes were identified belonging to 133 types. The majority of AMR genes and virulent genes were observed in Klebsiella pneumoniae (40%) and E. coli (71%), respectively. Sul2 was the most prevalent gene (%), followed by blaCTX-M-15 (5%), and aph (3'')-Ib (5%). fimH (6%), iutA (6%), and traT (5%) were the most prevalent virulent genes. The most common ESBL producer genes were blaCTX-M-15 (7%) and blaTEM-1B (6%) and the most common AmpC genes were blaCMY-2 (2%) and blaCMY-6 (1%). The highest carbapenemase genes were blaOXA-23 (3%), blaOXA-66 (3%), blaNDM-1 (2%), and blaOXA-1 (2%). In 82 (58%) of the sequenced strains, eight types of gene mutations were identified: parC (30%), gyrA (30%), ramR (1%), rpoB (1%), ompK37 (45%), acrR (43%), and ompK36 (43%) with a total of 220 antimicrobial gene mutations and 956-point mutations. However, 22 (2%) of the mutations were frameshift mutations and the rest 934 were point mutations. Among the 142 drug-resistant strains, 103 (73%) have plasmid mlst, and 108 (76%) have plasmid genes. Resistance to disinfectants like benzalkonium chloride 77(52%) and cetylpyridinium chloride 77(52%) had OqxA 41(29%), OqxB 41(29%), and qacE 49(35%) disinfectant-resistant genes. Like other AMR genes, NICU (B6) still has the leading prevalence of disinfectant resistance genes (22%) followed by C/W (9%) and pediatric hematology (D7) (8%). The study showed AMR has become a significant health hazard of bloodstream infection that most affects neonatal and ICU patients. The alarming result of this study was 7% PDR bacteria. The NICU by ESBL and SICU, General surgery, and caesarian section units by Carbapenemase producer strains were affected. A high burden of ESBL and Carbapenemase was seen in this study The predominant ESBL, MBLs, and Carbapenemase genes were blaCTX-M, blaNDM, and blaOXA-23 respectively. Nine novel drug-resistant bacteria strains were identified. Surgical and inserted medical instruments are the main drug-resistant bacteria transmission roots. Carbapenemase and MBL drug-resistant genes were associated with NICU, general surgery, SICU, and C/S unit which needs infection prevention standard implementation.Item Breast Cancer Subtypes, Associated Biomarkers, and the Involvement of Human Papilloma Virus in Ethiopian Population(Addis Ababa University, 2023-12-22) Esmael Besufikad; Adey Feleke; Tesfaye Sisay; Rawleigh HoweBreast cancer is the most common type of cancer in the world as well as in Ethiopia. Although research on breast cancer in Ethiopia has been conducted, none of them have evaluated breast cancer in multiple regions of the country, which is important considering Ethiopia’s enormous ethnic and genetic diversity. Hence, this study was carried out to evaluate the distribution of breast cancer subtypes and associated immune cell biomarkers, hormone receptors, matrix metalloproteinases (MMPs), and HPV genotypes in selected Ethiopian regions. A total of 227, 81, 58, and 120 formalin-fixed paraffin-embedded (FFPE) tissue blocks were collected for breast cancer subtyping, immune cell biomarkers analysis, MMP expression, and HPV genotyping, respectively. Immunohistochemistry (IHC) staining was performed for breast cancer subtyping based on estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), and Ki-67 proliferation markers, and for additional immune cell biomarker expression. RNA was extracted and quantitative reverse-transcription PCR was performed for MMP expression analysis. DNA was extracted from archived FFPE breast tissue specimens and target genes were amplified using PCR for HPV genotyping. SPSS Version 25 was used to enter and analyze data. For immune cell biomarkers and MMP results, GraphPad Prism version 8.0.0 was used for statistical analysis. A large percentage of breast cancers were found to have advanced clinical and pathologic features, such as substantial lymph node involvement, large tumor size, and high histological grade. The percentage of ER and PR-negative tumors were 48.3% and 53.2%, respectively. The IHC subtype distribution was 33.1% triple-negative (ER-, PR-, HER-2-) breast cancer, 27.6% luminal B ((ER+, PR+, HER-2- and Ki-67 ≥ 20%) or (ER+, PR+, and HER-2+)), 25.2% luminal A (ER+, PR+, HER-2- and Ki-67< 20%), and 14.1% HER-2- enriched (ER-, PR-, HER-2+). In multiple logistic regression analysis, grade III and HER- 2 positivity were associated with larger tumor size, and tumor size was also higher in samples from Southwestern Ethiopia (Jimma) as compared to Northern Ethiopia (Mekele). The MMP-11 expression levels were significantly higher in breast cancer cases than in benign breast tumors (P=0.012). The non-luminal (triple-negative and HER-2-enriched) breast cancer subtype had a higher percentage of stromal CD20+, intratumoral CD3+ tumor-infiltrating lymphocytes, and CD68+ tumor-associated macrophages than the luminal (Luminal A and Luminal B) subtype. The stromal programmed cell death ligand 1 (PD-L1) +, intratumoral CD3+ tumor-infiltrating lymphocytes, CD163+ tumor-associated macrophages, and PD-L1+ were also more commonly found in grade III breast cancer than in grade I and II breast cancer, respectively. Human papillomavirus was found in 20.6% of breast cancer patients and 29.6% of non-malignant breast tumors. Human papillomavirus infection was nearly 10-fold more common in ER-positive than ER-negative breast cancer. A considerably high prevalence of triple-negative breast cancer was reported in our study, demanding additional research that includes identifying genetic predisposition factors. A significant association was found between the breast cancer subtype and stromal CD20+, intratumoral CD3+ tumor-infiltrating lymphocytes, and CD68+ tumor-associated macrophages. The stromal PD-L1+, intratumoral CD3+ tumor-infiltrating lymphocytes, CD163+ tumor-associated macrophages, and PD-L1+ were also associated with tumor grade. Our findings suggest an important impact of MMPs in breast cancer pathophysiology, particularly MMP-11. This study also showed no proof of a link between HPV infection and breast cancer; however, the finding that HPV was more prevalent in breast tumors that were ER-positive than ER-negative warrants further attention.Item In Vitro Antipromastigote Activity of Decocted and Hydrodistilled Leaf Extracts of Clematis Hirsuta Against Leishmania Donovani and Leishmania Aethiopica(Addis Ababa University, 2024-01) Etsegenet Abebe; Gurja Belay; Tegenu Gelana; Solomon Mequanente; Yehenew AsmamawLeishmaniasis is one of the six most notable neglected tropical illnesses. The protozoan parasite known as Leishmania, which belongs to the family Trypanosomatidae, causes all three types of leishmaniasis in both the Old and New Worlds. The Food and drug administration approved liposomal amphotericin B (L-Amp B) as medication. Clematis hirsuta leaves and stem are used to treat leishmaniasis as locally known by the people of Debre Libanos monastery. This traditional claim is still untested, thus taking this evidence into consideration the current study was aimed at scientifically validating the antileishmanial potential of Clematis hirsuta by testing the in vitro antipromastigote activity of Clematis hirsuta decoction and hydrodistilled leaf extracts and to test for cytotoxicity. The extracts of Clematis hirsuta antipromastigote activities against the promastigotes of Leishmania aethiopica and Leishmania donovani at 100 μg/ml and their cytotoxic effects against human red blood cells were evaluated. The decoction extract of C. hirsuta showed 75.36 ± 1.47 % and 87.37 ± 0.39 % growth inhibition on L. aethiopica and L. donovani, respectively at 100 μg/ml. While the hydrodistilled extract of C. hirsuta showed 97.22 ± 0.02 % and 97.54 ± 0.07 % growth inhibition on L. aethiopica and L. donovani, respectively. The IC50 values of decocted extract were 0.01 μg/ml and 0.002 μg/ml against L. aethopica and L. donovani, respectively. While the IC50 values of hydrodistilled extracts were 0.39 μg/ml and 0.06 μg/ml against L. aethopica and L. donovani, respectively. On hemolysis assay, the decoction extract showed 18.18 ± 2.14 % hemolysis on red blood cells. While the hydrodistilled extract showed 57.57 ± 4.28 % hemolysis on red blood cells. The CC50 values of decocted extract and hydrodistilled extracts were >1000 μg/ml and 881.0 μg/ml on red blood cells respectively. On selectivity index values, the decocted extract had a SI >1000 on both Leishmania promastigotes. While the hydrodistilled extract had SI of 2258.97 on L. aethiopica and 14,683.3 on L. donovani. In conclusion, the decocted extract showed above 75% activity while the hydrodistilled extract showed above 97% activity against both Leishmania promastigotes. The extracts were less toxic against RBC as well. Both extracts were also selective against both Leishmania promastigotes. This suggested that these extracts were active against the parasite and validate the claims of its use against leishmaniasis. Future works are recommended to test antiamastigote activity.Item Screening of Endophytic Bacteria Isolated from Selected Plants in Hawassa for Production and Characterization Amylase(Addis Ababa University, 2024-06) Bethel Sitotaw; Asnake Desalegn; Fitsum TiguAmylases are crucial enzymes in the global industry, constituting 25% of enzyme production. Endophytes, known for producing bioactive compounds and enzymes, offer promise for industrial applications. The aim of the study was to screen amylase producing endophytic bacteria, to produce amylase under controlled laboratory conditions and characterize the enzyme. Purposive sampling was used to select the study areas as well as the plants screened for amylase producing endophytic bacteria. After collection the plant samples were washed and surface sterilized before isolation of the endophytic bacteria. The endophytic bacteria were isolated then screened for amylase production on starch agar media and purified. The pure isolates were characterized using morphological and biochemical test. Amylase was produced through submerged fermentation, and its production was optimized using different carbon and nitrogen sources, pH and temperatures. Ammonium sulphate precipitation and dialysis were used for partial purification and the partially purified enzyme was assayed for its activity using the dinitrosalicylic acid (DNS) method. Out of 60 endophytic bacteria isolated from the plants, 32 demonstrated amylase production. From these, 16 pure isolates were found to be the most efficient amylase producers and 7 best amylase producing isolates based on submerged fermentation. Among these top 16 producers, 12 were Gram-positive bacteria and 4 were Gramnegative bacteria.The 7 isolates were identified to species level by rRNA gene sequence analysis. Then 3 isolates were selected for further purification and characterization. Diameter (Mean ± SD) of clear zone on starch agar ranged from 4.48 ± 0.54 for isolate I10L2 from Enset to 11.53 ± 0.34 for isolate D10F2 from Datura. The highest (p < 0.05) amylase production was recorded for isolate D10L2 when glucose or starch was used as carbon source. The highest amylase production was recorded for all isolates in the presence of ammonium sulfate and the lowest in the presence of tryptone as nitrogen source. The optimum temperature for production of amylase was 30 OC, but differences in the optimum pH ranging from pH 5 to pH 8 were recorded based on the isolates preferences. After partial purification the highest enzyme activity was recorded at pH 7 and pH 9, 50 OC, and 0.5% and 1% calcium ion (Ca 2+) concentrations. The maximum activity in the presence of 1mM Ca2+ was about 16.5 U/mL. These findings highlight the potential of endophytic bacteria for amylase production. With further optimization and scale up, the amylase can be produced in large quantities and for specific applications.Item Evaluation of Beauveria Bassiana Isolates Against Varroa Destructor of Apis Mellifera Under Laboratory Condition(Addis Ababa University, 2024-04) Olyad Daniel; Asnake Desalegn; Yitbarek Woldehawariat; Zewdu ArarsoVarroa, Varroa destructor (Acari: Varroidae), is an ectoparasitic mite of honeybees, Apis mellifera. This parasite poses a substantial threat to the health, welfare and production of A. mellifera globally, including Ethiopia. Without chemical treatment, colony losses worldwide are common. However, resistance to synthetic chemicals in beekeeping is increasingly concerning. Entomopathogenic organisms provide an eco-friendly alternative to pesticides and can prevent resistance development. Therefore, isolates of Beauveria bassiana were tested for their pathogenicity against V. destructor and their negative effect on the brood and adult stages of the central highland honeybees of Ethiopia, A. mellifera. Varroa mites were immersed in 5 millilitres of a conidial suspension containing 1 x 108 conidia/mL of three different fungal isolates (APPRC-44BC, APPRC-27, and S#10H), as well as control solutions (0.05% Tween 80 and distilled water). The inoculated mites were placed on honeybee brood inside capped cells. Then, the infected brood combs were kept in an incubator at 33 ℃ and 60% relative humidity for ten days. The fungal isolates and control treatments were also applied to young adult workers and healthy brood to observe the effects of the treatments. Fungal isolate APPRC-44BC displayed the highest (73%) potential for killing varroa mite. All three isolates (APPRC-44BC, APPRC-27, and S#10H) were found to be highly efficient between the sixth and eighth days post-application, accounting for 96.8% to 100% of the fungal-induced deaths. Interestingly, treatment with B. bassiana isolates did not show a significant effect on brood emergence and the weight of newly merged adults. However, the treatments had significant effect on adult honeybee survival. It is clearly observed from these results that isolates of B. bassiana are potential bio-control agents against V. destructor. However, further studies are needed to evaluate the efficacy of this promising fungal isolate (APPRC-44BC) in honeybee colonies under field conditions, as well as to develop application methods that have minimal impact on adult honeybee survival.Item Morphological and Gdf9 Gene Polymorphism of Indigenous Goat Populations in Darra District(Addis Ababa University, 2023-12) Ejigayehu Siraj; Helen Nigussie; Gurja BelayGoats among the livestock species, are considered the most prolific ruminant which are adapt in different farming areas and agroecological zones in highland, subhumid, semiarid, and arid environments. Characterization of genetic resources is a prerrquesit to design sustainable genetic improvement program. Therefore the objective of the study was to assess morphological variations and GDF9 gene polymorphism in indigenous goat population in darra district North Shoa Zone of Oromia Regional State. A total of 336 goat population were selected from seven kebele those have single, twin, triple litter size using stratified purposeive sampling method. Body weight and linera body measurement were taken from 336 goats and blood samples were also collected from 150 goats only. Genomic DNA was extracted using salting out procedure. All samples were genotyped using GDF9 gene primer pairs. The most frequent coat color pattern observed was plain, spotted, red and brown were observed coat color types in the study area. The analysis of variance showed a significant variation (p < 0.05) in morphological traits among does in different agroecolgies,location with different litter size. Goats in highland agro ecology showed better body weight and linear body measurmetns compared to lowland agroecolgy. Goats having single litter size showed low value in most measuremtns compare to twin and triplet. However, there was no variation observed among goats of twin and triplet for except pelvic length. GDF9 locus was polymorphic in twin and triplet litter sizes and showed two alleles (462, 478) and three genotypes (462/462, 462/478 and 478/478) that segregated with different frequencies Genotype 462/462 had high frequence (0.47) followed by genotype 462/478(0.43) in twins where as genotype 462/478 (0.47) showed higher frequency followed by genotype 462/462 (0.38) in triplet. The expected hetrozygosity (HE) ranged from 0.43 in triplet to 0.47 in twin litter size. The closest genetic relationship was found between goats having twin and triplet (0.09), while goats of singleton and their triplet counterparts were more distant apart (0.16).Significant (p < 0.05) and positive association of GDF9 genotypes with litter size and linear body measurements were observed. Morphological variation and genetic variation at GDF9 locus could be an opportunity to design cost effective breeding strategy. However,the current result is prilimnary, large nuber size at different location should be considred to subsatatiate the current result.Item Physico-Chemical and Microbiological Analysis of Honey and Tej collected from Central Ethiopia and Production of Tej Using Starter Culture(Addis Ababa University, 2024-06) Frehiwot Dagima; Mogessie AshenafiTej is the most diverse traditional fermented alcoholic beverage of Ethiopia. It is prepared from honey, water and leaves of Gesho (Rhamnusprinoides). In our country, Tej is commonly fermented by natural micro flora. The aim of this study was to investigate the physicochemical, proximate and microbiological properties of honey and Tej from central part of Ethiopia and its surrounding and production of Tej by using starter culture. pH. specific gravity,moisture content, ash, acidity,electrical conductivity, Hydroxyl methyl furfural, sugarsand alcohol contents are the main physicochemical and proximate parameters analyzed in honey and Tej samples. Aerobicspore-forming bacteria, Aerobic Mesophilic bacteria, Enterbacteriaceae,Yeastand Mouldsand Total coliforms were the microorganisms assessed for the Tej samples. Atotal of 20 honey and 30 Tej samples were collected.The mean values of pH and Acidity of Tej and honey rangedfrom3.45-4.20and1.0-2.7,JorTejandfrom2.96-4.45and17.87-52.15forhoney, respectively,The mean moisture and ash contents of Tejandhoneyvariedfrom86.7-92.7and0-0.2,/01'Tej,80.01-84.75 and 0-0.3 for honey respectively. The mean value of ECofTej ranged from 0.44-0.76andthevaluesof honey ranged from 0.27-0.92.The HMF of honey ranged from 0.0-1.05,and the mean of alcohol content ranged from 8.62-14.49 for the Tej samples. Yeasts were the dominant microorganisms found in the Tej and they were not defected in honey samples. The yeast counts were ranged from 5.60-8.00Cfu/g. Aerobic spore-forming bacteria (ASFB),Total coliform and Enterbacteriaceae were not detected Based on their growth performance under different physiological stressestenLAB and ten yeast isolates were screened. The Yeast isolates were tentatively identified into four species level, Saccharomyces cerevisie, Saccharomyces daireness, Debaryomycescarsonii. Both the LAB and yeast isolates were combined in different proportions to formulate starter cultures for the production of Tej. Ten formulations were made in different proportions based on their compatibility of the isolates. Using Four Yeast and Ten LAB-Yeast starter culture formulations, Tej was prepared under controlled fermentation conditions. The overall sensory acceptability analysis showed that formulate.F#5,F#2,and F#7 were the best mixed starter cultures for Tej preparation as compared with the control.However, further molecular identification of the isolates into species level and investigation of the microbial dynamics of Tej is recommended for future use of these isolates, The result for physicochemical and proximate analysis were aligned with national and international standards.Item Physico-Chemical and Microbiological Analysis of Honey and Tej Collected from Central Ethiopia and Production of Tej Using Starter Culture(Addis Ababa University, 2024-06) Frehiwot Dagima; Mogessie AshenafiTej is the most diverse traditional fermented alcoholic beverage ofEthiopia. It ispreparedfrom honey, waterand leaves of Gesho (Rhamnusprinoides). In our country, Tej iscommonly fermented bynatural microflora. The aimof this study wasto investigate thephysicochemical,proximate and microbiological properties of honeyand Tejfrom centralpart of Ethiopiaandits surrounding and production ofTejbyusingstarterculture.pH. specific gravity,moisture content, ash, acidity,electrical conductivity, Hydroxyl methyl furfural, sugarsand alcoholcontents arethe main physicochemical andproximate parameters analyzed in honey and Tejsamples. Aerobicspore-forming bacteria, Aerobic Mesophilic bacteria, Enterbacteriaceae,Yeastand Mouldsand Total coliforms were themicroorganisms assessed fortheTejsamples.Atotal of 20 honey and 30 Tej samples were collected.The meanvalues ofpHandAcidityofTejandhoney rangedfrom3.45-4.20and1.0-2.7,JorTejandfrom2.96-4.45and17.87-52.15forhoney, respectively,Themeanmoistureandashcontentsof Tejandhoneyvariedfrom86.7-92.7and0-0.2,/01'Tej,80.01-84.75and0-0.3forhoneyrespectively. ThemeanvalueofECofTejrangedfrom0.44-0.76andthevaluesofhoneyrangedfrom0.27-0.92.TheHMFofhoney rangedfrom0.0-1.05,andthemeanofalcoholcontentrangedfrom8.62-14.49fortheTej samples.Yeastswerethedominantmicroorganismsfoundin theTejandtheywerenotdefectedinhoneysamples.The yeast countswere rangedfrom 5.60-8.00Cfu/g. Aerobic spore-forming bacteria (ASFB),TotalcoliformandEnterbacteriaceaewere not detected Basedon theirgrowthperformanceunderdifferent physiologicalstressestenLAB and ten yeast isolateswerescreened. The Yeast isolates were tentativelyidentified intofour species level, Saccharomyces cerevisie, Saccharomyces daireness,Debaryomycescarsonii. BoththeLAB andyeast isolates were combined in differentproportions toformulate starter culturesforthe production of Tej. Tenformulations were made in different proportions based ontheir compatibility of theisolates. Using Four Yeast and Ten LAB-Yeast starter cultureformulations, Tej was prepared under controlled fermentation conditions. The overallsensoryacceptability analysisshowedthatformulate.F#5,F#2,andF#7werethebestmixedstarter cultures forTejpreparation as comparedwiththe control.However, further molecular identification of theisolatesintospecies level and investigation ofthemicrobialdynamics ofTejisrecommendedforfutureuseofthese isolates, The result forphysicochemical and proximateanalysiswere alignedwith nationaland internationalstandards.