Microbial, Cellular and Molecular Biology
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Item Screening of Endophytic Bacteria Isolated from Selected Plants in Hawassa for the Production and Characterization of Amylase(Addis Ababa University, 2024-06) Bethel Sitotaw; Asnake Desalegn; Fitsum Tigu (PhD)Amylases 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 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 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 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 BelayEthiopia 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 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 PetrosClassificahon 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 (≤ 0.6x109 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 Ethno-Botany, Genetic Diversity, Micro-Propagation and Nutritional Profiling of Enset [Ensete Ventricosum (Welw.) Cheesman] Landraces from Central Ethiopia(Addis Ababa University, 2024-06) Tesfaye Dilebo; Tileye FeyissaEnset [Ensete ventricosum (Welw.) Cheesman] is a perennial, multipurpose crop that serves as a staple or co-staple food for about 25 million people in Ethiopia. Despite its significant importance and the existing knowledge about diverse enset landraces and their external morphology and complex internal anatomical features, there are limited documented studies on this crop. Furthermore, the ethnobotany of ethnolinguistic communities in the country, culturally linked to the use and management of enset, is a complex and under-researched area. Therefore,this study was focused on the wealth of farmers’ indigenous knowledge on farm-level enset diversity, distribution, and selection patterns, the local landrace identification criteria, and nomenclatural system with the evaluation of the extent of genetic diversity and population structure, further developing an efficient micro-propagation protocol and identifying the nutritional and anti-nutritional contents of selected landraces. The study and sample collections were performed in the Hadiya, Kembata, Gurage, and Silte administrative zones of Ethiopia. A total of 240 enset farmers were surveyed using semi-structured interviews for ethnobotanical research and documentation of indigenous knowledge. The Shannon-Weaver, Simpson, Pielou, and Sorenson’s similarity indices were also used to evaluate the diversity and similarity of the enset landraces. To evaluate the extent of genetic diversity and population structure, a total of 147 individual leaf samples were collected from the Hadiya, Kembata, Gurage, and Silte zones and the Areka Agricultural Research Center, and the analysis was computed by using 12 simple sequence repeat (SSR) markers. For the in vitro propagation approach, about 1.0 cm long shoot tips were cultured on MS medium gelled with 5%–10% bulla and supplemented with 1–6 mg/l BAP, either separately or in combination with IAA. Regarding the proximate, the Association of Official Analytical Chemists (AOAC) standard methods were applied. Minerals, phytate, tannin and Oxalate contents were determined using the different models of the spectrophotometer methods and the standard procedures. A total of 282 farmer-named enset landraces have been identified, ranging from 2 to 32 on individual homegardens. The Hadiya Zone had the highest number of enset landraces (86), while the Silte Zone had the lowest number (57). The results of the Shannon diversity index and Simpson’s 1-D diversity index indicated the presence of high enset diversity in the study zones. The Sorenson’s similarity index ranged from 0.24 to 0.73, sharing 16–47 landraces in common. Of the 282 landraces, 210 (74.5%) were recorded in more than one zone, whereas 72 (25.5%) had a narrow distribution being recorded in a single zone. The local names of some enset landraces indicate their uniqueness in morphological traits, place of origin, agronomic features, and quality attributes of their end products. The 12 SSR markers result shows a total of 289 alleles, ranging from 12 to 41 alleles per locus. The polymorphism information content (PIC) for each locus varied from 0.86 to 0.95. The number of effective alleles ranged from 5.13 to 11.79. The expected and observed heterozygosity showed average values of 0.85 and 0.84, respectively. Among the six populations, the wild-growing population had the highest percentage of polymorphic loci (100%). AMOVA attributed 89% of the genetic variation to intra-population and only 11% to among populations. The UPGMA and principal coordinates indicate three major groups. The 8% (w/v) of enset bulla was ideal and provided significant figures in the number and length of shoots and roots per shoot when compared with 0.6% (w/v) agar-gelled MS media. MS medium containing 2.0–3.0 mg/l BAP was the appropriate concentration for in vitro shoot induction and growth. The 4.0 mg/l BAP alone and 5.0 mg/l BAP in combination with 1.0 mg/l IAA were suitable for multiple shoot induction, whereas 2.0 mg/l IBA and 1.0 mg/NAA separately were found to be the optimum concentrations for root induction and development. The proximate composition (%) ranged in moisture content from 68.2–79.4, crude protein (2.43–11.90), crude fat (0.61–0.89), crude fiber (2.42–4.11), and total ash (2.01–4.60), while the total carbohydrates came to 80.89–89.92, and gross energy was 369.96–385.12 kcal/100 g. The mineral concentrations (mg/100 g) ranged from 22.46–49.74 for calcium, 28.51–86.56 for potassium, and lower for magnesium, phosphorus, sodium, iron, and zinc on a dry weight basis. The anti-nutritional contents (mg/100 g) for phytate, tannin, and oxalate ranged from 221.75–276.12, 27.97–113.74, and 5.69–9.10, respectively. Except for phytate×calcium to zinc, and oxalate to calcium, the molar ratios were above the standard values. Overall, the information gained from this study would be useful for improving programs and conservation strategies for the enset crop, which enhances Ethiopia's sustained food security.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 Assessment of Bovine Mastitis in Lactating Cows, and Characterization of Staphylococcus Aureus from the Cows and Dairy Products in West Showa Zone, Ethiopia(Addis Ababa University, 2024-06) Negassa Feyissa; Asnake DesalegnMastitis, to which Staphylococcus aureus is one of the main causative agents, is the costly mammary disease which adversely affects dairy industry worldwide in general and in developing countries in particular. S. aureus is a Gram-positive coccus resides on the skin and mucus linings, and associated with various diseases in animal and human. The virulence factors of S. aureus associated with disease causing potential and/or survive in the harsh melena include different enzymes and toxin productions, antibiotic resistance, and survival in dry conditions and relatively high concentration of salt. A cross-sectional study with objectives of determining the prevalence of mastitis in lactating cows, and assessing the prevalence and virulence factors of S. aureus isolated from the lactating cows, and bulk milk, and milk products (yogurt and cheese) was, conducted from May 2020 to March 2021 in West Showa Zone of Oromia, central Ethiopia. Stratified random sampling method was applied to select the districts. The PAs, the farm (herds), and individual sampling units were selected randomly. Mastitis was diagnosed physically by observations and palpation for clinical mastitis, and by California Mastitis Test (CMT) method for subclinical mastitis. Teat milk from the lactating cows, bulk milk, yogurt, and cheese samples were collected for bacterial isolation. Farmers and milk product retailers were interviewed using structured questionnaire to assess the hygienic milk collection and storage practices in the study area. The biological samples were aseptically collected, labeled, and were transported under cold chain to the Zoonosis and Food Safety Research Laboratory of Ambo University for bacterial isolation. S. aureus was selectively isolated on a manitol salt agar at 37°C for 24 hours, and presumptively identified by growth and rapid manitol fermentation within 24 hours, Gram staining and observation under microscope, and catalase and coagulase tests. Finally, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-ToF) test was used to confirm the species. The enzyme production ability of the isolates was determined using heated plasma agar for staphylokinase, butterfat-supplemented brain-heart infusion agar for lipase, and skimmed milk agar for protease. The enterotoxin genes, resistant genes (blaZ, and mecA), and thermo nuclease gene (nuc) were assessed using a specific primer-based conventional polymerase chain reaction (PCR). In addition, the antibiotic susceptibility pattern of the isolates was determined using the disc diffusion method on Muller Hinton agar. The data obtained from the field assessment, laboratory experiment, and questionnaire surveys were analyzed using SPSS version 20 and R-statistical software. . The data were summarized using frequencies‘ and percentage, and presented in graphs and tables. The risk-factors associated with the prevalence of mastitis and S. aureus , and virulence factors of S. aureus, were analysed and interpreted by using chi-square test, logistic regression, t-test, and ANOVA at 95% confidence interval (C.I.) with p<0.05 significance levels. Accordingly, 258 lactating cows were diagnosed for mastitis and 97 (37.6%) of them were mastitis-positive. The prevalence of mastaitis was found significantly increased with the advancement of lactation stage of the cows, in those milked with unwashed hands and unwashed udders before milking, and in cows infested by ticks when compared with early lactation stage, those milked with washed hands and udder before milking, and tick free cows respectively (p<0.05). Subclinical mastitis was significantly more prevalent than clinical mastitis (p<0.05). The result of the study also disclosed that 94 (15.36%) of 612 samples were positive for S. aureus. The isolation rate of S. aureus was significantly higher in mastitis-positive cows (35.05%) than in mastitis-negative cows (5.59%) (p<0.05). The prevalence of S. aureus was also significantly higher in cows reared in farms with large number of lactating cows and tick-infested cows than in cows of farms with small number of lactating cows and in tick free cows, respectively (p<0.05). In addition, 354 bulk milk and milk product samples were collected and the bacterium was significantly more frequent in bulk milk (20.3%) and cheese (14.9%) than in yogurt (4.8%). Moreover, 71 (77.2%), 51 (55.4%), and 62 (67.4%) of 92 S.aureus isolates were positive for protease, staphylokinase and lipase respectively. The PCR result revealed that the S.aureus isolates harbored sea, seb, and tsst-1 genes at rate of 12/24 (50%), 3/24 (12.5%), and 4/24 (16.6%) respectively. The S. aureus isolates were resistant to penicillin(100%), tetracycline (100%), oxy-tetracycline (100%), amoxicillin (100%), ampicillin (83.3%), and to norfloxacin (75%). Of the 92 isolates tested 12(13%) were found resistant to oxacillin (1 μg) and were considered MRSA. The PCR test of the resistant genes (mecA and blaZ) revealed that 10/44 (22.73%) were mecA positive; but none of them harbored the blaZ gene. In addition, all (100%) of the 34 isolates tested for the thermonuclease (nuc) gene were positive to the gene. The questionnaire survey also indicated that all of the 140 respondents milked the animals manually. More than 50% of them have not washed their hands before milking; have not used the udder drying clothes; and/or used it for more than one cows; used plastic utensils for collecting and storing milk and milk products; and have not smoked the utensils. Therefore, it is concluded that mastitis was prevalent in the study area, and S. aureus might be the major cause of the disease. In addition, the milk and fermented milk products might not be safe for human consumption. Moreover, the unhygienic milking practices and tick infestation were one of the factors playing role in the prevalence of mastitis in the study area. The milk collection and storage practices might exacerbate the cross contamination of the dairy products by pathogens from the miller‘s hand, utensils and/or the environment so that the source of the S. aureus could be the animals, the handlers or the utensils. The presence of the enterotoxin genes in the bacterium is indicative for the food intoxications caused by the toxins. Furthermore, different enzymes producing ability of the isolates, summed up with resistance to commonly used antibiotics revealed that the S. aureus isolates are highly virulent to cause serious and incurable infections. The availability of the resistant genes (mecA) particularly in the isolates from milk and milk products can be evident for the possible transfer of the gene to other bacterium if co-infection occurs or to the enterobactriacae found in the GIT. Therefore, other mastitis causing agents and their antibiogram characteristics and virulence factors, and the types of MRSA distributed in the area should be studied. In addition, in order to prevent and control the disease and cross contamination of the dairy product, the animal and dairy product handlers should be trained about the hygienic milking practices and food handling methods.Item Microbial Quality and Safety Assessment of Fresh Lettuce in Addis Ababa, Ethiopia(Addis Ababa University, 2025-02-27) Yeabsera Damtew; Fitsum TiguLettuce is high in water content (94-95%) and low in calories. It's also high in vitamins, minerals, and bioactive substances including polyphenols, carotenoids, and chlorophyll, all of which have health benefits. Food-borne illness outbreaks linked to the consumption of ready-to-eat vegetables are on the rise. but the recent information about the microbial loads and safety aspects of fresh lettuce are lacking. In this study, the microbial quality and safety assessment of fresh lettuce in Addis Ababa, Ethiopia were investigated. The general objective was to assess the microbial quality and safety and identify the major food-borne pathogens from fresh lettuce samples collected from selected marketplaces in Addis Ababa. A stratified random sampling was used to select 110 vegetable sellers in Addis Ababa, Ethiopia. Detection of hygiene indicators microorganisms: total coliforms, aerobic mesophilic bacteria (AMB), yeast, mold and Enterococci, E. coli, and other food-borne pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, were analyzed from 110 fresh lettuce samples. The study reveals that the fresh lettuce samples collected from selected marketplaces in Addis Ababa were contaminated with various microorganisms, including total coliform, Staphylococci, Fecal streptococci and E. coli this indicates the poor hygienic conditions of the study area. As a confirmatory test, 33 positive representative isolates after the biochemical test were taken and detected by polymerase chain reaction (PCR). Among the analyzed samples, only 2 samples Addis Ketema and Lideta sub cities were positive for E. coli and P. aeruginosa, respectively. This contamination poses a potential biological health hazard to general consumers, as they are at risk of contracting food-borne infections. The findings underscore the urgent need for improved hygiene and safety measures in the production and handling of lettuce to safeguard consumers from potential biological health risks in Addis Ababa.Item Isolation and Characterization of Dichlorodiphenyltrichloroethane (DDT) Degrading Fungi Isolated from Agro-Industrial Effluent and Farm Soils(Addis Ababa University, 2025-02) Girma Ebsa; Tesfaye AlemuDichlorodiphenyltrichloroethane (DDT) is an environmentally hazardous synthetic compound that is resistant to usage. Despite being illegal in most countries, it is used as a pesticide to fight malaria in most malarial zone part of Ethiopia. The main objective of this study is to screen, characterize, and evaluate potential DDT-degrading fungi and their synergetic interaction effects for mycoremediation purposes. The composite of 150 soil and effluent samples was collected from Addis Ababa and East Shewa Zone, mainly Ziway and Koka. Fungal isolation and screening were performed using a serial dilution on potato dextrose agar growth media. MALDI-TOF MS technology was used for fungal identification. Fungal biomass production and sporulation capacity were examined and optimized using a Box-Behnken experimental design. The potential DDTtolerant fungi were studied based on growth factor optimization. Gas Chromatograph-Electron Capture Detector technology was used for the DDT degradation study. Fungal identification results revealed that the finally selected isolates, AS1 and T1, were Asppergillus niger and Trichoderma koningii, respectively. The optimization results confirmed that the co-inoculated isolates AS1T1 had a maximum biomass (1.01±0.16 g) and spore count (5.74±0.37 log spore/mL) and were selected as possible DDT-degrading fungi. The GC-ECD result analysis revealed that fungal-cocultured A. niger and T. koningii in DDT-amended liquid medium were able to degrade DDT into its metabolites Dichlorodiphenyldichloroethane (DDD) and Dichlorodiphenyldichloroethylene (DDE). The results also revealed that 99.5–99.99% of DDT and its metabolites degraded from initial concentrations of 1750, 3500, 5250, and 7000 ppm. The co-inoculated fungi A. niger and T. koningii are promising candidates for the removal of DDT and its metabolites from polluted environments.Item Genetic Diversity and Drug Resistance Pattern of Mycobacterium Species Among Smear Negative Pulmonary Tuberculosis Patients in Addis Ababa, Ethiopia(Addis Ababa University, 2025-01) Alem Alemayehu; Beyene Petros; Rawleigh Howe; Liya WassieTuberculosis (TB) is being treated empirically in about 38% of patients globally due to difficulty in detecting the Mycobacterium tuberculosis complex (MTBC) bacilli with the routine laboratory diagnostic tests, including smear microscopy, GeneXpert and culture. As a result, the situation with smear negative pulmonary tuberculosis (SNPTB) has remained challenging, leading to diagnostic delays and making monitoring treatment outcomes difficult. However, there is scarce information on the actual burden, drug resistance pattern and the strains of MTBC involved in SNPTB. Therefore, this study aimed to evaluate the genetic diversity and drug resistance pattern of MTBC isolates recovered SNPTB patients. A health facility-based case control study was conducted in Addis Ababa, Ethiopia, involving 313 pulmonary TB patients (173 SNPTB and 140 Smear positive PTB (SPPTB)). Laboratory data was generated by collecting sputum specimens from consenting study participant and acid fast staining, GeneXpert and culture were done at Armauer Hansen Research Institute (AHRI) laboratory. Molecular analysis, spoligotyping and whole genome sequencing (WGS) as well as phenotypic drug susceptibility test were done for culture grown MTBC isolates. The revised online MTBC molecular marker database SITVIT2 and appropriate bioinformatics tools were applied to assign lineages and sublineages of MTBC strains. Structured questionnaires were used to assess TB related factors, clinical and imaging findings associated among SNPTB and SPPTB patients. SPSS version 25 and Stata version 17 software were used for data analysis and a P-value < 0.05 was considered statistically significant. Of the 173 SNPTB patients 75 were culture positive. The spoligotyping and WGS analysis of MTBC isolates identified four major MTBC lineages including lineage 4 (Euro-American), lineage 3 (East-African-Asian), lineage 1 (Indo-Oceanic) and lineage7 (Ethiopian) which had 80.2%, 16.7%, 2.4% and 0.8% proportion, respectively. Lineage 3 MTBC strains were higher among SNPTB patients compared to SPPTB patients while the proportion of lineage 4 MTBC strain was higher among SPPTB than SNPTB (P-value <0.05). A higher proportion of MTBC isolates from SNPTB 16.7% were resistant to one or more first-line anti-TB drugs than SPPTB 12.9% (P-value >0.05). MDR-TB was detected from single SNPTB patients 5.6% (1/18) by both phenotypic and molecular methods whereas none in SPPTB patients (0/33) (P-value >0.05). Although, overall clinical presentations of SNPTB patients resemble those seen in SPPTB patients, prior history of TB was strongly associated with SNPTB (P-value <0.05). There was a significant difference in MTBC strain distribution among SNPTB and among SPPTB patients, which may be responsible for difficulty in MTBC laboratory detection that may need further advanced analysis of sequenced isolates. There was also primary drug resistant TB among culture positive SNPTB patients, which would be otherwise missed by current national protocols. An additional study with larger sample size and more sensitive TB detection approach is recommended for better understanding of SNPTB.Item Screening and Characterization of Thermostable Alpha- Amylase from Bacteria Isolated from Hot Environments(Addis Ababa University, 2025-02) Weldesemayat Getachew; Fitsum Tigu; Asnake Desalegn (PhD)Alpha-amylase is one of the most important enzymes for undergoing in starch saccharification and liquefaction to results simple sugar. Despite there were numerous resources to isolate thermostable alpha-amylase producing bacteria however little research was conducted in our country. The objective of this study was to screen, and characterize thermostable alpha-amylase producing bacteria. A totally of 35 samples were gathered from selected hot environments around Addis Ababa. A total of 188 bacterial isolates were screened for alpha- amylase production through starch hydrolysis test. Of which, 161 isolates were obtain to be positive for starch hydrolysis test. Sixty-six isolates with amylolytic index (AI ≥ 1.5+0.3 mm) were selected for submerged state fermentation and the enzyme assay was conducted through DNSA method. Based on their fermentation performances, 6 isolates, FBW2-3A, FHW5-1A, KS1-1A, SJS1-4A, KHW2-2A and SAS1-2A were selected for further amylase production optimization process. All the six bacterial isolates were phenotypically and molecularly characterized into species level. They belonged to Bacillus cabrialesii (FBW2-3A), Acinetobacter haemolyticus (FHW5-1A), Enterobacter quasiharmachei (SJS1-4A), Bacillus cereus (KHW2-2A) and Pseudomonas plecoglossicida (SAS1-2A) while KS1-1A isolates was not identified. The effect of temperature, pH, incubation period, carbon and nitrogen sources, starch and salt concentration, metal ions and inoculum size were optimized based on one factor at a time approach. For all isolates the optimal growth temperature, pH and incubation time was obtained at 50oC, pH 7 and 24 h, respectively. Crude alpha-amylase was optimally active at 55oC with pH 7. The crude enzyme was thermally stable above 90% at 55oC for 30 min incubation. Partially purified amylase from Bacillus cabrialesii (FBW2-3A) has a specific activity of 10.12U/mg, which is 1.8-fold than the crude enzyme with molecular weight lies approximately 20kDa. The food industrial efficiency of the alpha amylase was investigation by applying of crude alpha-amylase into wheat flour fermentation and it showed a good dough raising capacity.Item Assaying of Alpha Amylase Extracted from Mesophilic Bacteria from Bishoftu Lakes for Detergent Applications(Addis Ababa University, 2025-02) Misgana Anbessa; Fitsum Tigu (PhD); Asnake Desalegn (PhD)Alpha amylase is a biological catalyst which breakdown large molecules of starch to smaller unit. It has many functions in industries like food, fermentation, detergent, textile, and pharmaceuticals. This study aimed to isolate, characterize, and evaluate alpha-amylase-producing mesophilic bacteria from Bishoftu lakes for potential application in detergent formulation. The samples were isolated using serial dilution and nutrient agar media. Hydrolysis method was employed to screen all isolates for their potential of amylase production by starch plate agar method. Overall, 110 isolates were tested as positive for amylase activity among 169 isolates. Based on amylolytic index (AI), 60 isolates were used for submerged state fermentation and 9 most potent amylase producing isolates were subjected for further analysis. The impact of temperature, pH, incubation time, carbon source, organic and inorganic nitrogen sources, metal ions, inoculum size, and various concentrations of starch, and sodium chloride on the isolates were performed. The maximum alpha-amylase production of all the 9 isolates was observed at 40ºC in 24 h of incubation time, pH 7, glucose, tryptone, beef extract, ammonium chloride, zinc sulphate, potassium phosphate, 1.5% sodium chloride concentration using basal media. The biochemical tests like methyl red, Voges Proskaur, citrate, urease and catalase tests were conducted. A total of 9 isolates were identified. 7 isolates were grouped as B. cereus and the rest as, A. rivipollensis and K. pasteurii based on 16S rRNA, gyrB and elongation factor Tu (tuf) gene sequencing. A. rivipollensis (G2W1) recorded 4.5 ± 0.1 U/mL after applying all optimum growth conditions, which was the highest one. The second was K. pasteurii (G3W1) (4.3 ± 0.4 U/mL) and the third was B. cereus (B1W1) (4.1 ± 0.1 U/mL). The optimum conditions for the crude enzyme activity were 45ºC, pH 7, 15 min of reaction time, Ca2+, and in which 5 ± 0.04 U/mL of enzyme activity was recorded by A. rivipollensis. Ca2+ was the best enhancer among metal ion as cofactors. Partially purified enzyme has a molecular weight of 50 kDa, with specific activity of 10.1 U/mg. The effectiveness of alpha amylase was checked by preparing a detergent and the soap made by this enzyme has successfully cleaned the pieces of cloth that were purposively contaminated by potato waste.Item Diversity and Phytobeneficial Properties of Indigenous Root Nodule Bacteria and Arbuscular Mycorrhizal Fungi Associated with Erythrina Species and their Effect on Growth of the Host Plant under Greenhouse Conditions(Addis Ababa University, 2021-04-09) Berza, Belay; Assefa, Fassil (Professor)Erythrina is a leguminous tree used in agro-forestry practices in the southern and southwestern Ethiopia. The legume-rhizobium symbiosis provides N to plants, while Legume-AMF symbiosis enhances P availability. Despite the crucial agro-forestry attributes, there were scarcities of information on species diversity of arbuscular mycorrhizal fungi (AMF) associated to E. brucei, genetic and functional diversity, and eco-physiological stress tolerance traits of bacteria isolated from the root nodules of E. abyssinica. The objectives of this study were to; 1) determine AMF species diversity and richness associated with Erythina brucei; 2) the genetic and functional diversity and eco-physiological stress tolerance traits of bacteria isolated from the root nodules of E.abyssinica ;3) evaluate inorganic phosphate solubilizing efficiency and multiple phytobeneficial properties of root nodule bacteria associated with E. abyssinica; 4) evaluate the symbiotic effectiveness of Bradyrhizobium species, and evaluate the effects of multiple inoculation of the consortia of microorganisms on Erythrina abyssinica growth, nodulation, and shoot TN and P contents in greenhouse condition. Soil samples were collected from different E.spp growing areas involved various land use types and trap cultures were established. AMF spore extraction and species identification was done from soils obtained from the rhizosphere of E. brucei. The root nodule bacteria of E. abyssinica were obtained by plant infection method. The genetic diversities were studied using Amplified ribosomal DNA restriction analysis (ARDRA). The nifH was gene screened and the taxonomic position was determined using 16S rRNA sequence analysis. Plant growth promoting traits were also evaluated and selected microbial inputs were studied for their effects on E. abyssinica growth, nodulation, nitrogen fixation and uptake P in the greenhouse condition. Eleven AMF genera and 33 species were recovered. The ARDRA grouped the bacterial isolates in to twelve clusters at 70 % similarity level. NifH gene was amplified in 15 (50%) of the root nodule bacteria the 16S rRNA sequence analysis grouped the bacterial in to nine genera (species): Bradyrhizobium (n=3), Paenibacillus (n=2), Bacillus (n=2), Staphylococcus (n=2), Enterobacter (n=2), Achromobacter (n=1), Acinetobacter (n=3), Gluconobacter (n=4) and Stenotrophomonas (n=2). Achrmobacter, Acinetobacter, Bacillus, Gluconobacter, Paenibacillus, Staphylococcus and Stenotrophomonas are the first reports from E. brucei root nodules. In addition, Bradyrhizobium cajani and Bradyrhizobium cytisi are the first reports from E. abyssinica root nodules and from Ethiopia as well. The 61.9%, 76.2%, 47.6%, 33.3% and 19.04% of the isolates were inorganic phosphate solubilizers, IAA, NH3, HCN and chitinase producers respectively. The maximium shoot length and dry weight improvements (140%) and (267%) were recorded by multiple inoculations involved B. shewense (AU27) + Glomus sp.1(AMF1) + Acaulospora sp.1 (AMF2) + A. soli (AU4). Similarily, the maximum shoot total nitrogen improvement (260%) was recorded in treatment received B. shewense (AU27) + A. soli (AU4) inoculation; whereas the highest shoot P improvement (1200%) was recorded by B. shewense (AU27) + Glomus sp.1 (AMF1) inoculation. AMF species richness was affected by land use types and geographic locations. Phylogenetically and functionally diverse bacteria were inhabited in the root nodules of E. brucei. Multiple inoculations enhanced E. abyssinica growth and development, biomass production, nitrogen fixation and multiple inoculations also improved phosphorus uptake by the host plant. Synergistic interaction was observed among the microsymbionts and macrosymbiont. More studies are required to explore the rhizosphere of E. abyssinica and the performance of the microbial inputs which exhibited potential performance in greenhouse needs evaluation in the field condition.Item Genetic and Physiological Diversities of Bacteria from the Rhizosphere of Woody Plants Grown on Soil from Girar Jarso District and their Effect on the Growth and Establishment of Tree Seedlings in North Shewa Zone Ethiopia(Addis Ababa University, 2020-04-19) Getahun, Alemayehu; Muleta, Diriba (PhD); Assefa, Fassil (Professor)Land degradation (LD) is one of the major problems the planet earth has been facing. It has severely affected 1.9 billion hectares and decreased ecosystem services by 60%. In Eastern Africa, particularly in the northern highlands of Ethiopia, 1 billion tons of topsoil are lost annually. Thus, the land becomes bare, nutrient-depleted, water-stressed and abandoned by the community. Hence, it needs urgent rehabilitation with the application of eco-friendly microbes and organic amendments (OAs) through the exploitation of different types of trees/shrubs. Therefore, the overall objective of the study was to rehabilitate degraded habitat through the application of OAs and phytobeneficial bacteria for the establishment of multipurpose trees. Soil samples were collected from nine random corners at depth of 30 cm for soil physicochemical analysis before and after OAs. Six different treatments (biochar, compost, manure, mixed, bacterial inoculation (BI), and control) were considered at a 1:1 ratio of OAs per pot. Application BI was done for field trials during transplanting and quarterly for a year. Six plots measuring 41 m x 4 m were established in completely randomized block design and assigned at the random block for the field trial. Following OAs, microbial counts were done for one year every month. Bacteria were isolated from the rhizosphere of Acacia and Juniperus. The primary selection of isolates was based on drought stress (DS) tolerance and phosphate solubilization, other stress, and plant growth-promoting traits. The potential isolates were subjected to carbohydrate and amino acid utilization tests, BOX PCR, and 16S rRNA profiling. DS tolerant, phosphate solubilizer strains with multiple plant growth-promoting traits were chosen for Acacia seeds germination and field application alone and in combination with OAs. Plant growth parameters and their survival rate at each amended plot were assessed. There is a significant increase in soil pH (5.69-8.13), CEC (43.78-49.98 cmolc/kg), OM (2.43-3.91%), total nitrogen (0.13-0.76%), and available P (18.9-26.31 ppm) following OAs compared the control. Combined treatment had the largest effect on cover crops biomass with 3.43 g, 4.54 g, 0.7 g, 2.07g in alfalfa, grass pea, and control respectively p ≤ 0.05. The C and N utilization revealed metabolic versatility of the strains (14.29 to 100%). Ochrobactrum, Pseudomonas, and Klebsiella spp expressed remarkable metabolic versatilities. BOX-PCR showed greater genetic diversity and confirmed by Simpson’s Index of Diversity (0.883) took the leading position with Bacillus species. The 16S rRNA genes sequence showed 21.92% Firmicutes and 78.08% Proteobacteria with Pseudomonas 23% and Ochrobactrum 21% dominant species. Out of 73 isolates, 10 (14%) were highly tolerant of 40% polyethylene glycol. All the isolates can grow in wider ranges of pH (5-9), temperature (15-45°C). The inoculated bacterial strains significantly p ≤ 0.05 increased root, shoot length, and dry biomass of acacia. According to solubilization index (SI) 45% isolates were classified as high and medium phosphate solubilizers with 195 to 373 μg/mL. The maximum P and IAA were produced by Pseudomonas FB-49 (373 and 659.07μg/mL), respectively. The highest (100%) seed germination caused by Pseudomonas BS-26 and Pseudomonas FB-49. There is a significant difference in microbial counts following OAs compared to the control. The greatest counts in bacteria, actinomycetes, and fungal (21.66, 2.29, 0.82 x 105 CFU g-1) of soil, respectively in the combined amended plot. There was a significant increase in stem height, girths, and branch numbers in amended plots relative to the control. The survival rate was observed in apple (80%) followed by acacia (66%) and prunus (51%). The survival is in the order of BI x OAs > BI > biochar > compost > manure > control amended soil. This study concluded that degraded land could be rehabilitated with cheap OAs, potential bacterial strains, and bring multipurpose tree establishment with greater survival rate and best performance.Item Evaluation of Phyto Beneficial Traits of Indigenous Phosphate Solubilizing Bacteria and Fungi as Microbial Inoculants for Enhancing Growth and Production of Coffee (Coffea Arabica) Under Greenhouse and Field Conditions in Jimma South West of Ethiopia(Addis Ababa University, 2021-12-17) Abafita, Reshid; Assefa, Fassil (PhD); Muleta, Diriba (PhD)Exploitation of phosphate solubilizing bacteria (PSB) and fungi as microbial inoculants is known to promote plant growth through the supply of plant nutrients and supression of pathogens. In view of this, the present investigation was planned to assess the phytobeneficial traits of phosphate solublizing bacterial and fungal isolates recovered from coffee (Coffee arabica) and vermicompost to determine their potential in growth promotion of coffee seedlings under low input agriculture.. The microbes were isolated and purified following standard methods. The selected isolates were investigated for their plant growth promoting properties, eco-physiological tolerance under laboratory conditions, and further tested under greenhouse and nursery experiments. The greenhouse and nursery experiments were conducted with completely randomized design (CRD) in three (3) replications per treatments. Thus, a total of 154 bacteria and 72 fungi isolates were recovered from which twelve potent bacterial and nine fungal isolates were selected and investigated for their plant growth promoting properties. Among the twelve bacterial isolates, three of them were tentatively identified to the genera of Pseudomonas (RCHVCB1) and Bacillus (RScB1.19 and RMaB2.11), and showed significant potential to solubilize Ca3 (PO4)2 and posessed several phytobeneficial traits, viz, indole acetic acid, NH3, HCN productions and N-fixing ability. They also exhibited remarkable tolerance to ecophysiological factors such as heavy metal, acidity and salinity, and inherent antibiotic resistance (IAR). Similarly, three fungal isolates with superior phosphate solubilizeation ability were characterized and identified as genera of Penicillium (RSCF1.19) and Aspergillus (RCHVCF2 and RLVCF2). During co-culture, RSCF1.19 (Penicillium sp.) slightly inhibited the test pathogen, Fusarium xyloriodes. The bacterial (RCHVCB1, RScB1.19, RMaB2.11) and fungal isolates (RSCF1.19, RCHVCF2, RLVCF2) enhanced rate of coffee seed germination under laboratory conditions and promoted coffee seedlings growth under glasshouse conditions. The results of inoculated seeds showed significant (p≤0.05) differences in germination rate and vigor index compared to the control. Isolates RScB1.19, RMaB2.11+RSCF1.19 and RMaB2.11 + RLVCF2 showed high germination rate (20.59%) over the control (13.33%). Moreover, a single inoculation of RLVCF2, RSCF1.19 and co-inoculation of RMaB2.11 with RLVCF2 also showed significant (p≤0.05) mean root length (1.31 cm) and mean shoot length (1.48 cm) over the control. Under greenhouse conditions, single inoculation of RSCF1.19+phosphate and dual inoculation of RSCF1.19 and RCHVCB1 in the presence of inorganic phosphate fertilizer led to significantly higher plant height, root length, stem girth, leaf number, leaf area, fresh and dry weights. Due to high pH value of the potting medium (vermicompost alkaline pH-pH>7.5), all the treatments combined with vermicompost showed suppressive effect and no any seedlings were emerged at all. Under nursery conditions, co-inoculation of RSCF1.19 with three bacterial isolates (RCHVCB1, RScB1.19, and RMaB2.11) in combination with inorganic phosphate led to significantly increase the tested growth parameters. Similar increase in growth attributes was observed in both single and dual inoculations due to vermicompost used compared with both positive and negative controls. Higher NPK-uptake was observed in a combination of bioinoculants and inorganic phosphate fertilizer compared to the positive and negative control. In general, inoculation of RSCF1.19 and RLVCF2 isolates to coffee 74110 variety combined with inorganic phosphate fertilizer resulted in good vigor and healthier coffee seedlings (RSCF1.19, 34.42%) and (RLVCF2, 37.09%) when compared to control (28.49%). Therefore, both RSCF1.19 and RLVCF2 fungal isolates could be used as bioinoculants after field trials in coffee 74110 variety productions.Item Detection of Major Enteropathogens Associated with Calf Diarrhea in Dairy Farms of Afar Region Ethiopia(Addis Ababa University, 2021-10-21) Abera, Eskedar; Desalegn, Asnake (PhD); Kebede, Nigatu (Professor)Diarrhea is one of the most common causes of sickness and mortality among newborn calves. It has serious financial and animal welfare indications in both dairy and beef herds. Diarrhea in a newborn calf is one of the most common diseases in young animals, causing huge economic and productivity losses to livestock industry worldwide. Dairy farming is a growing cattle production system in Ethiopia. However, morbidity and mortality of calves are among the factors that have been hindering success of dairy industry. Multiple enteric pathogens such as virus, bacteria and parasite are the most common causes of calf diarrhea. However, various facets of diarrheal disease caused by rotavirus, coronavirus, E. coli K99 and Cryptosporidium parvum infections in calves in Afar region are inadequately understood. A Crossectional study was carried out from February 2020 to April 2021 with the aim of prevalence and associated risk factors of rotavirus, coronavirus, E. coli K99 and Cryptosporidium parvum infections in calves from selected districts of Afar national regional state, in north east Ethiopia. A total of 176 fecal samples of 0 up to 6 months old diarrheic calves were collected by purposive sampling. Samples were tested for those four pathogens using a commercially available enzyme-linked immunosorbent assay (ELISA). In addition, different factors were measured the level of association between variables. Out of the 176 samples tested 59 were positive for at least one pathogen by ELISA. Positivity rates for each pathogen were, coronavirus 45 (25.5%), Cryptosporidium parvum 29 (16.5%), rotavirus 18 (10.2%) and K99 enterotoxigenic E.coli 11(6.25%). From positive viral samples of ELISA test 38 were propagated in Madin Darby bovine kidney cells. After 3 following passage, progressive cytopathic effect (CPE) i.e. rounding, detachment as well as demolition of mono-layer cell of all samples was observed. The study sit, time of first colostrum feeding, and duration of colostrum feeding were significantly associated with the occurrence of rotavirus, coronavirus, Cryptosporidium parvum and E. coli and this are indicative of the need for strict prevention and control mechanisms such as practicing early colostrum feeding in newborn calves.Item Genetic Characterization and Estimation of Genotype by Environment Interaction of Ethiopian Sesame (Sesamum Indicum L.) Germplasm(Addis Ababa University, 2021-07-02) Tesfaye, Tewodros; Tesfaye, Kassahun (PhD)Sesame is one of the major oil crops that has great economic importance for the country. In Ethiopia, sesame is among the foremost important oil crops both in terms of area coverage and total national annual production. However the crop suffers from low productivity due to biotic and abiotic stresses. Therefore, the present study conducted in different sets to generate information that can be used to design the future breeding program of sesame in the country. The first set of experiments was to study the morphological and molecular genetic diversity of the sesame germplasm collected from Ethiopia and other countries (Asian and other African countries). The same genotypes planted at three locations for phenotyping and genotyping using the two high throughput diversity array technology (DArT) markers (silicoDArT and SNP). Further to understand the impact of different putative genes Genome-wide association study of yield-related traits using 2997 SNPs in two environments was performed. The second set of experiments was conducted in 19 environments to assess the performance and stability of sesame varieties, and to characterize sesame growing environments in Ethiopia. Based on morphological characterization, genotypes showed wide variability for most morphological traits, except for plant growth type, leaf glands, anther filament color, and anther connective tip gland. High heritability combined with high genetic advance was recorded for plant height, primary branch, days to flower initiation, days to 50% flowering, pod bearing zone, seed yield per plant, and bacterial blight reaction indicating the potential of improving the population through a direct selection for these traits. Grain yield showed a significant and positive genotypic correlation with plant height, the number of capsules per plant, and pod bearing zone, the magnitudes of the positive genetic correlation suggest that the selection by those characters produces a significant increase in grain yield. Genetic divergence using Mahalanobis D2 statistics was computed, and the genotype lines were grouped into six different clusters. Clustering was not associated with the geographical distribution; instead, genotypes were grouped mainly based on morphological differences. The maximum inter-cluster distance was observed between clusters IV and VI (D2 =342.56, followed by clusters I, and VI (D2 =217.9783). Maximum genetic recombination and variation in the subsequent generation are expected from crosses that involve parents from the clusters characterized by maximum distances. The genetic diversity analysis showed that the average nucleotide diversity of the panel was 0.14. Considering the genotypes based on their geographical origin, Africa collections (0.21) as a whole without Ethiopian collection was more diverse than Asia and when further portioned Africa, North Africa (0.23) collection was more diverse than others, but at the continent level, Asia (0.17) was more diverse than Africa (0.14). The genetic distance among the sesame populations ranged from 0.015 to 0.394, with an average of 0.165. The structure analysis divided the panel into four hypothetical ancestral populations and 21 genotypes were clustered as an admixture. Under Genome-wide association study (GWAS) a total of 21 significant SNPs with 7 yield-related traits in two environments were identified and, these explaining the phenotypic variation ranged from 7.02 (DF) to 16.11% (CAPL), with an average of 9.76%, suggesting a moderate contribution to the traits. All significant loci found in LG 2, 6, and 11 associated with capsule length except one associated with the physiological period. The significant loci found in LG 3, 7, and 8 associated with a physiological period (Days to flower initiation, Days to 50% flowering, and Days to physiological maturity) except one associated with capsule length. Dissecting genetic control of flowering time and maturity is importance to foster sesame breeding and to develop new varieties able to adapt to changing climatic conditions. Indeed, flowering time and maturity strongly affect yield and plant adaptation ability. Since several favorable alleles detected in this study have not yet been intensively selected, our GWAS results will assist in incorporating further useful alleles into the elite sesame germplasm for a seed yield increase in the future. Based on the genotype x environment interaction study the test locations were divided into six groups. Humera, Banat, and Tach Armacho were highly discriminating and representative in the first, the second, and the third group respectively, and were identified as a core test site in that group. While Alemaya, Worer, and Mender67 were identified as the only test site in groups four, five, and six. The core testing sites identified would be used to facilitate the identification of superior sesame varieties and to reduce testing costs in the country. Environment Tach Armacho in 2017/18 and 2018/19 were close to the ideal environment. The GGE biplot analysis identified genotype G2 (setit-1) as the “ideal” genotype and among the highest mean seed yield. Setit-1 considered the most stable across variable environments.Item Estimation of Breeding Parameters within and between Malt Barley (Hordeum Vulgare L) Crosses using Phenotypic Traits and Kasp SNP Markers(Addis Ababa University, 2021-12-31) Tadesse, Endeshaw; Tesfaye, Kassahun (PhD)The genetic variation in a breeding program is created by crossing genetically divergent parents. The resulting genetic variation between and within crosses is the determining factor for the offspring’s performance, which is defined as the level of compliance of the offspring with preset breeding goals. Estimation of breeding parameters plays a key role in crop improvement. The aim of this study was to (i) estimate and compare mid-parent value and the cross mean (ii) estimate and compare the variance between means of crosses (σ²c) and segregation variance of recombinant inbred line within crosses (σ²g) (iii) estimate correlation among the traits measured and their heritability (iv) estimate and compare Rogers Distances (RD) of parental lines and their correlation to the segregation variance within crosses, and (v) estimate usefulness of crosses. 900, F4:5 recombinant inbred lines randomly derived from 30 crosses were evaluated at two locations in modified split plot p-rep design whereas the parental where genotyped using SNP-KASP markers. The variance component analysis revealed significant (P <0.05) genetic variation among parents, crosses and RIL for almost all traits. Based on generation mean analysis, the means of recombinant inbred line did not deviate significantly from the means of parental lines. The range of σ²g for the individual crosses was very high for days to heading, days to maturity, plant height, thousand kernel weight and grain protein concentration and for these traits the respective standard deviation were high. Heritability for parents ranged from 49.50 % (malt extract) - 93.60 % (plant height), heritability for crosses ranged from 29.52 % (grain protein concentration) - 87.0% (days to maturity), whereas heritability for RIL was lowest with 27.40% (beta-glucan) - 73.60% for thousand kernel weight. Significant (P<0.01) genotypic correlations with high impact for practical breeding were found between malting traits. Significant (P<0.01) regression of cross mean on mid-parent value were obtained for all traits demonstrating that cross means can accurately be predicted from mid parent values and selection among crosses at an early stage of line development is highly effective. Further, based on the usefulness criteria, 16 outperforming crosses were identified that surpass Planet as the actual leading malt variety. Diversity analysis revealed an average RD between lines was 0.46 with a range of 0.32- 0.64 and homogeneity within the 17 parental lines varied from 71-100%. The dendrogram grouped the lines into three clusters. Importantly, the high malting European lines were grouped together with some of the ICARDA and Ethiopian lines Correlations of RD to σ²g ranged from -0.19 to 0.34 and were not significantly deviating from zero for all traits except DH, hence, RD proved to be not predictive for σ²g. In this study the two variances, σ²c and σ²g, proved to be significantly deviating from zero for almost all traits; hence breeder can exploit both of them for selection. Generally, differences between generation means (Parental lines vs. RIL) were small across all crosses. But when grouping into crosses with and without Planet as a parental line, deviation between parent and RIL were larger possibly due to epistastic effects. Hence breeder should estimate breeding values rather than genetic values of parental lines. The use of mid-parent value and usefulness for cross selection were found to be promising. The national barley breeding program should exploit this result by starting with a large number of initial crosses and reducing them to the most promising crosses. Some heterogeneity was observed within the lines. When originating from technical mixture this should be considered as a wake-up call for the breeder to closely follow maintenance breeding. RD based on KASP markers was not predictive for σ²g; hence further research will be required by improving accuracy of σ²g estimate and linkage disequilibrium between markers and quantitative trait loci (QTL). Over all, the large diversity revealed among lines indicates the potential that the genotypes have to improve the productivity and quality of the crop and the uses of molecular markers can benefit the breeding program in order to have an effective breeding program by selecting diverse parental genotypes with complementary gene action.Item Characterization of Ethiopian Durum Wheat Landraces and Cultivars for Processing Quality Using Phenotypic Traits and Simple Sequence Repeat (SSR) Markers(Addis Ababa University, 2021-09-02) Dagnaw, Temesgen; Tesfaye, Kassahun (PhD); Haileselassie, Teklehaimanot (PhD); Geleta, Mulatu (PhD)Durum wheat (Triticum turgidum L. ssp. durum Desf.) is getting attention in terms of production and area coverage at the global level. Its unique characteristics to produce pasta and related end-products increased the preference for durum wheat by millers and processors. Ethiopia is considered as a center of diversity for durum wheat. However, in Ethiopia, durum wheat is facing serious genetic erosion due to the expansion of teff and bread wheat, and the cultivation of exotic durum wheat materials. The genetic resource of the Ethiopian durum wheat genotypes has been limitedly explored for its processing quality attributes at the level of both phenotypic and molecular diversity. Therefore, the present study was aimed to assess the genetic diversity and evaluate the processing quality attributes of Ethiopian durum wheat genotypes using phenotypic traits and SSR markers. The field experiment was conducted at two locations (Sinana in Bale zone and Chefe Donsa in East Shewa zone) to assess the phenotypic diversity. The phenotypic characterization of genotypes showed a wide range of variability for most quantitative and qualitative traits. The combined analysis of variance (ANOVA) showed highly significant (P<0.01) variations among genotypes for the majority of the traits studied. Gluten content (GL) and grain yield (GY) showed high and intermediate heritability, respectively, combined with moderate genetic advance, and grain protein content (GPC) showed intermediate heritability combined with low genetic advance. Both, GPC and GL showed a significant and negative correlation with GY. Cluster analysis based on quantitative traits grouped the genotypes into 5 major clusters. The first four principal components (PCs) explained 64% of the total variation. Based on qualitative traits, high genetic variation was observed among genotypes. Correspondence analysis discriminated cultivars from other populations of landraces. Finally, 104 best-qualified genotypes for processing quality traits were selected and assessed using quality traits associated 14 SSR markers, which had high mean polymorphic information content (PIC) of 0.56 and gene diversity (0.61). High levels of genetic diversity was obtained among all populations (I = 0.86; He = 0.46). Analysis of molecular variance (AMOVA) revealed high genetic variation within populations (88.35%) and among populations (11.65%). The Neighbor-joining (NJ) clustering and principal coordinate analysis (PCoA) grouped the genotypes into three major clusters. In addition, Bayesian model-based population structure analysis revealed two major genetic groups. Overall, this study revealed high genetic diversity among Ethiopian durum wheat landraces and cultivars, and these genotypes can be used to identify the best genotype/s for processing quality attributes and subsequent use in the Ethiopian durum wheat improvement programs.