Microbial, Cellular and Molecular Biology

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Actinomycetes From Unexplored Environmental Niches in Ethiopia and Their Biotechnological Potentials for Antimicrobial Compound Production
(Addis Ababa University, 2018-06-04) Kibret Moges; Abate Dawit (PhD); Zotchev Sergey B Professer); Rollinger Judith M. (Professer)
Actinomycetes have a widely recognized potential for the production of significant bioactive compounds. The major aim of this study was to isolate, screen and evaluate the biotechnological potential of selected actinomycete isolates particularily for antimicrobial compound production using standard bioassays methods, LC-MS, high resolution mass spectrometry (HR-MS) and NMR techniques. It consists of six distinct chapters. In the first chapter, general introduction, statement of the problem and major objectives are presented. The second chapter deals with the review of related literatures. The rest four chapters (3-6) are the experimental sections of the work. Hence, the third chapter concentrated on the isolation, screening, bioactivity detection and phylogenetic analysis of promising actinomycetes capable of producing bioactive secondary metabolites from various unexplored niche habitats in Ethiopia. Among the 416 isolates screened for bioactivities, 101 (24%) isolates were inhibiting the growth of C. albicans, and 88 (21%) isolates were inhibiting both C. albicans (ATCC 62376) and C. neoformans (clinical isolate). Ten isolates having considerable activities were chosen for further investigation and taxonomic identification studies. The polyphasic identification results of these isolates found to be in consistent with the genus Streptomyces described in Bregay’s manual of systematic bacteriology. Identification of the isolates have been verified by the analysis of the 16s rRNA gene sequence. The phylogenetic relationships of the isolates to type strains and best matches based on BLAST search were inferred using the Maximum Likelihood algorithm in MEGA 7 software and confirmed that all the isolates belong to genus Streptomyces. The fourth chapter deals with the cultivation of five promising isolates namely Ac-029, Ac-125, Rv-355, Ac-464 and Go-475 for bioactive secondary metabolite production and subsequently evaluation of SSF process parameters on metabolite yield. Depending on the types of the isolates, variations were observed ii in optimal fermentation process parameters on bioactive secondary metabolite production. It was demonstrated that wheat bran in the presence of supplementary nutrients, an initial moisture content of 65%, a pH value of 7.5, incubation temperature of 30 oC, an inoculum size of 3x107 CFU/mL and incubation period of 12 days were the optimal SSF conditions for most of the isolates studied. The fifth chapter focused on antimicrobial potential of Streptomyces sp. Rv-355 cultivated in submerged culture. In its bioactivity profile, Streptomyces sp. Rv-355 produced antimicrobial compounds with wider spectrum of activities against yeasts, Gram positive and Gram negative bacterial pathogens. It was found that biomass production and bioactivity profiles of Streptomyces sp. Rv- 355 are positively correlated. Bioactivity guided analysis of the crude extract from Streptomyces sp. Rv-355 using TLC, column chromatography, HPLC, LC-MS showed the presence of potential compounds. The partially purified extract showed MIC values of 50μg/mL against Candida albicans and 100μg/mL against Bacillus subtilis. The result is found to be a prelude for further analysis of the crude extract from Rv-355 using HR-MS, and NMR methods. The sixth chapter was targeted on the bioactivity guided identification and structural elucidation of members of benz[a]anthraquinone antibiotics, 8-O-methyltetrangomycin and 8-O-methyltetrangulol from Streptomyces sp. Go-475 extracts using LC-MS, HR-MS/MS and 1H NMR 13C NMR methods. Streptomyces sp. Go-475 displayed potent activity against both yeasts and Gram-positive bacteria with MIC values of the crude extracts 100μg/mL and 50μg/mL against Candida albicans ATCC62376 and Bacillus subtilis ATCC6633 respectively. The analysis revealed that Streptomyces sp. Go-475 is able to produce at least three known secondary metabolites (4-Methoxy-1(3H)-isobenzofuranone, 3-Phenylpropionic acid or 1, 2- Benzenediol and Dehydrocineromycin B) that were not detected in the SmF extract. However, betaine was detected in both SSF and SmF extracts of this isolate. Two important anti-bacterial iii compounds were purified from methanol extract of Streptomyces sp. Go-475 and their structures were elucidated by NMR and HR-MS/MS as 8-O-methyltetrangomycin and 8-Omethyltetrangulol. Besides, many potentially novel metabolites were detected, the majority of which were produced in SSF method. The findings enable us to conclude that Streptomyces sp. Go-475 and other isolates from Ethiopian soil have the capacity to produce potentially new antifungal secondary metabolites and warrant further investigations. The results also proved that SSF as promising economical and best option to produce potential bioactive secondary metabolites from Streptomyces spp. The genome sequence of Streptomyces sp. Go-475 was obtained using a hybrid assembly approach of high quality Illumina short read and low quality Oxford Nanopore long read data. The complete linear chromosome of 8,570,609 bp, featuring a G+C content of 71.96%, contains 7,571 predicted coding sequences, 83 t(m)RNA genes, and six rrn operons. Analysis of the genome for secondary metabolite biosynthesis gene clusters allowed us to connect certain clusters with experimentally confirmed molecules. The findings also verified great potential of Streptomyces sp. Go-475 for the production of chemically diverse secondary metabolites.
The Aetiological Causes of Tuberculous Lymphadenitis in Butajira, Ethiopia
(Addis Ababa University, 2001-06) Habte, Abebe; Olobo, Josph (PhD); Harboe, Morten (Professor); Lakew, Mekuria (PhD)
The utility of MPB70 antigen in serodiagnosis of Mtuberculosis complex infection and the polymerase chain reaction (PCR) for rapid identification of the causative agent if cervical lymphadenitis were investigated. The PCR assay was based on detecting a 506-bp DNA segment belonging to the alpha 32 kDa antigen (85B) common in the genus Mycobacterium, a 984-bp DNA segment belonging to the insertion sequence IS 6110 specific for the M tuberculosis complex, and a 185-bp pncA gene segment at position 169 allele-specific for genetic differentiation of Mtuberculosis from M bovis. For the ELISA purpose, sera from 25 tuberculous lymphadenitis (TBLN), 14 non-tuberculous lymphadenitis (NTBLN), 11 pulmonary tuberculosis (PTB) patients and 10 healthy control (HC) subjects were tested. For PCR, in 39 clinically diagnosed tuberculous lymphadenitis patients, fine needle aspirates (FNA) were processed and tested. Of these, which 14 were considered as non-tuberculous lymphadenitis by fine needle aspirate cytology (FNAC). Of the 39 clinically diagnosed TBLN, 27 (69%) were positive by ELISA. When this was compared with the assumed gold standard (FNAC), it showed 64% sensitivity, 21% specificity, 49% efficiency, 59% positive predictive value and 25% negative predictive value. With PCR, 29 (74%) of 39 were positive, showing 72% sensitivity, 21 % specificity, 54% efficiency, 62% positive predictive value and 30% negative predictive value. When it was compared with FNAC.Of the 29 PCR positive for genus Mycobacterium and Mtuberculosis complex, 18(62%) of them were identified as M tuberculosis, 3 (10%) of them were M. bovis and 8 8%)of them were found co-amplifying M.tuberculosis and M. bovis. These data indicated that both serodiagnosis with MPB70 antigen of the M.tuberculosis complex and peR assay are useful for rapid identification of tuberculous lymphadenitis and better management of patients.
Aflatoxins (B1, B2, G1 And G2) and Aflatoxigenic Fungi In Export Standard White and Red Sesame Seeds (Sesamum Indicum L.) Grown In Humera and Wollega, Ethiopia
(Addis Ababa University, 2017-06-03) Makiso, Markos; Zewdu, Ashagrie (PhD)
The oilseed sector is one of the fastest growing sectors in Ethiopia, both in terms of its foreign exchange earnings and as source of income for millions of Ethiopians. Oilseed crops are recognized to be potentially suitable substrates for the production of aflatoxins by aflatoxigenic fungi. This study aimed to evaluate Knowledge attitude and practice (KAP) of sesame exporters about aflatoxin contamination, analyze thousand seed weight, moisture, crude fat and peroxide value of white and red sesame collected from Humera and Wollega varieties. Determining the level of aflatoxins of white and red Ethiopian sesame seeds. Compare the level of aflatoxins between white and red Humera and Wollega varieties. Isolate and identify aflatoxigenic fungi from sesame seeds. A total of 27 sesame samples were collected from Ethiopian Agricultural commodities warehouse service enterprise, located in Addis Ababa. Semi-structured questionnaires used for knowledge, attitude and practice survey of sesame exporters and Thousand seed weight, moisture, crude fat and peroxide value of sesame seeds have been done according to American oil chemists society (AOCS) method. Analysis of aflatoxins was done by using HPLC with solid phase extraction clean up method. Isolation and identification of aflatoxigenic fungi has been done by culturing sesame samples on Potato dextrose agar (PDA) media. Sixty percent of exporters doesn't have any idea about aflatoxins and all of them cannot make aflatoxins test for sesame seed before export. Thousand seed weight, moisture, crude fat and peroxide value were ranged 2.89-3.22g/1000 seed, 3.33-4.99 %, 46.75-57.75% and 2-8.2 meq/kg, respectively. Aflatoxins detected from 93 % of samples in the range of (0.44 - 48.28 ng/g). Average content of AFB1, AFB2, AFG1, AFG2 and total aflatoxins are 0.83 ng/g, 6.82 ng/g, 17.25 ng/g, 1.17 ng/g and 26.07 ng/g, respectively. AFB1 and AFG2 detected below European union (EU) maximum limit for individual aflatoxin (2 ng/g). AFB2, AFG1 and total aflatoxins content is above the permissible limit set by EU for both individual and total aflatoxins (15 ng/g). Red sesame variety indicates relatively higher level of aflatoxins contamination as compared with white Humera and white Wollega sesame. Aspergillus flavus, A. parasiticus and A. niger isolates identified. Based on the finding, the contamination level may affect national economy.
Age and growth determination of tilapia, Oreochromis niloticus L. (Pisces: Cichlidae) in some lakes in Ethiopia
(Addis Ababa University, 1998-12) Admassu Demeke; Mengistou Seyoum (PhD)
Tilapia, Oreochromis niloticus L. is the most important fish in Ethiopia. Knowledge on age and growth of the fish is essential for fisheries management, and for further detailed studies. In this study, age and growth of O. niloticus in Lakes Zwa\, Langeno and Chamo was studied from microzones and macrozones in otoliths. The usefulness of otoliths to determine age was established by examining the existence of regularly formed translucent and opaque macrozones. The effect of subtle changes in temperature on body growth and on otolith macrozone formation was also experimentally tested. The growth of the fish in various lakes was also compared. Two translucent macrozones, each associated with biannulus (a type of annulus formed twice a year), were fOlmed each year in otoliths of O. niloticus in Lake Zwai. Biannulus formation was connected to fluctuations in temperature, quality and quantity of food, and to intensive spawning activity. Translucent macrozone was fOlmed in otoliths, and somatic tissue growth was slow, in tilapia that were transferred from 240C to slightly lower temperature treatments. Fish that were transferred from 240 C to higher temperatures grew rapidly, and more opaque material was deposited in their otoliths. At the high test temperatures, translucent macrozone was formed in otoliths of fish that had dpening or dpe gonads. O. niloticus in Lake Zwai has two spawning peaks, hence two major annual recruitment cohorts. Fish from the two cohorts were discriminated and age was determined from the number of biannulus in otoliths by relating median hatch-dates with the dates of capture. Ages determined in this manner (macrozonal ages) were highly correlated (r2 = 0.85) with microzonal ages. Growth of juvenile O. niloticus in Lakes Chamo, Zwai and Langeno was described by Gompertz model fitted to length and microzonal age data. Growth was most rapid in Lake Chamo, intennediate in Lake Zwai and slowest in Lake Langeno. Rapid growth of juvenile O. niloticus in Lake Chamo was attributed mainly to warm temperature and better food quality in this lake. The growth of adult O. niloticus in Lakes Zwai, Awassa and Haiq was described by von Bertalanffy model fitted to mean length and macrozonal age data. Growth rate was most rapid in Lake Haiq, intermediate in Awassa and least in Zwai. Rapid growth in Haiq was attdbuted to better quality food in this lake.
Agro-Morphological and Molecular Genetic Diversity, and Cytogenetic Analysis of Ethiopian Potato [Plectranthus Edulis (Vatke) Agnew] From Ethiopia
(Addis Ababa University, 2018-06-03) Gadissa, Fekadu; Tesfaye, Kassahun (PhD); Geleta, Mulatu (PhD)
Agro-morphological and Molecular Genetic Diversity, and Cytogenetic Analysis of Ethiopian Potato [Plectranthus edulis (Vatke) Agnew] from Ethiopia Fekadu Gadissa Addis Ababa University, 2018 Ethiopian potato syno. Ethiopian dinich [Plectranthus edulis (Vatke) Agnew] (Lamiaceae) is one of the ancient annual edible tuber crops, originating in Ethiopia. The crop is commonly cultivated by smallholder farmers around homesteads in the highland and semi-highland areas, usually for household consumption and rarely for marketing. In spite of its’ wide economic importances, the crop is neglected by research and development community and currently it is at risk of total extinction. Hence, this study was conducted with the main aims of assessing agro-morphological and molecular (EST-SSRs) markers-based extents of genetic diversity analysis as well as chromosome number and ploidy level determination using 174 accessions from diverse agro-ecologies in Ethiopia. For agro-morphological diversity analysis, the experimental samples were tested at Ambo and Holeta agricultural research centres, using an alpha lattice design at the locations and three blocks/replication followed by appropriate management practices. Agronomic and morphological traits-based data were collected on twenty-eight (12 qualitative and 16 quantitative) traits at the right growth stage and analysed using SAS v9.0, MINITAB® v14.13 and FigTree v1.4.3. Cytogenetic characterization was also carried out using very young root tips generated from soil covered stem rings, followed by appropriate pre-treatment, fixation and maceration. For molecular genetic diversity analysis, genomic DNA was extracted form silcagel dried young leaves collected from 287 plants (1-3 plants per accessions) following CTAB protocol. EST-SSRs marker were designed from Plectranthus barbatus cDNA sequences deposited in the GenBank, followed by PCR amplification, capillary electrophoresis, peak identification, and scoring. The scored allele size data were analysed for polymorphism, diversity indices and genetic relationship and structure using windows compatible applications. The agro-morphological traits considered showed varied morphotypes in all of the leaf (four in leaf color, three in leaf arrangement, three in leaf shape), stem (three in stem color, two in each of stem spot and stem spot colors) and tuber (four in tuber skin color, three in each of tuber texture, tuber shape and tuber hair) characterstics. Similarly, the traits revealed a wide range of variability in mean performance (minimum range of 1.39 – 2.13 cm observed in tuber diameter and maximum range of 112.90 – 165.10 xvii days observed in days to 50% flower initiation) and variance components among and within the accessions. Similarly, the mean square of all the traits showed a highly significant (P< 0.001) variation among the tested accessions. Such wide variation suggests the presence of variability which can be exploited through selection. Several of the traits showed a slightly greater or nearly equal phenotypic coefficient of variation (PCV) to that of genotypic coefficients of variation (GCV), suggesting larger contribution of the genotypic effect for phenotypic expression of such characters and hence, phenotypic values-based selection for the traits may be effective. High estimates of GCV (>23%) coupled with high estimate of heritability (Hb%) (>94%) and high genetic advance as a percent of mean (GAM) (>46) were revealed in tuber weight per hill, number of primary branches per plant, number of tubers per hill, and number of plants per hill indicating the importance of such traits for selection in Ethiopian potato improvement programs. The significantly positive phenotypic, and genotypic correlation in tuber weight per hill and number of tubers per hill with each other and several other traits as well as their negative phenotypic correlation with some other traits indicates the direction of selection. Moreover, the significantly higher absolute magnitudes in genotypic correlation compared to their corresponding phenotypic correlation suggest the genetic base of those traits. Cytogenetic characterization revealed a very smaller sized metaphase chromosome with a count of 2n = c.56 and hepta- or octa-ploidy was speculated on the bases of basic chromosome number reports (x = 8 or 7) for the species of genus Plectranthus and other members of the Lamiaceae family. Such chromosome count and ploidy level report could serve as a baseline information in selection and crosshybridization of Ethiopian potato with other closely related species. In total, twenty new polymorphic expressed sequence tag based simple sequence repeat (EST-SSRs) markers have successfully been developed and used in genetic diversity analyses. The marker detected a total of 128 alleles (6.4 alleles per locus) over the entire loci and populations with effective number of alleles ranging from 1.06 - 3.17 (an average of 1.67). The marker showed an overall highest (94.17%) percent polymorphism, and extents of PIC in the range of slightly informative to highly informative suggesting the potential of those developed markers as a valuable genetic tool and resource to evaluate the extent of genetic diversity and population structure of not only Ethiopian potato but also various other species within the Lamiaceae family. The ranges and levels of mean observed heterozygosity (0.33 – 0.429), Shanon’s information index (0.523 – 0.663), and Nei’s gene diversity (0.307 – 0.384) across loci showed a medium degree of variation among the populations which is a direct reflection of sharing of most of the alleles among the populations that partly resulted from high overall gene flow (Nm=18.29). Comparatively, Wenbera (Wen), Wolaita Sodo (WSo), Hadiya and Kambata-Tembaro (HKT) and Southwest Shewa (SwSh) populations could be considered as Ethiopian potato diversification and in-situ conservation sites. xviii Hierarchical analysis of molecular variance (AMOVA) showed significant but low population differentiation with at most 3% of the total variation in each of the groupings, such as among the populations, among geographic regions, and among regions of accessions. Likewise, cluster analysis in all the cases and STRUCTURE analyses did not group the populations into sharply distinct clusters, which could be attributed to historical and contemporary gene flow and/or the reproductive biology of the crop. In conclusion, this study has wider implications in bringing such a ‘super-neglected’ crop to the scientific agenda and thus, opens up the door to its improvement and conservation. However, it is important to exhaust all areas and regions in the country and more number of SSRs or other up-to-date molecular marker systems to come up with more accurate level of genetic diversity estimates.
Agro-morphological and Molecular Genetic Diversity, and Cytogenetic Analysis of Ethiopian Potato [Plectranthus edulis (Vatke) Agnew] from Ethiopia
(Addis Ababa University, 2018-06-03) Gadissa, Fekadu; Tesfaye, Kassahun (PhD)
Agro-morphological and Molecular Genetic Diversity, and Cytogenetic Analysis of Ethiopian Potato [Plectranthus edulis (Vatke) Agnew] from Ethiopia Fekadu Gadissa Addis Ababa University, 2018 Ethiopian potato syno. Ethiopian dinich [Plectranthus edulis (Vatke) Agnew] (Lamiaceae) is one of the ancient annual edible tuber crops, originating in Ethiopia. The crop is commonly cultivated by smallholder farmers around homesteads in the highland and semi-highland areas, usually for household consumption and rarely for marketing. In spite of its’ wide economic importances, the crop is neglected by research and development community and currently it is at risk of total extinction. Hence, this study was conducted with the main aims of assessing agro-morphological and molecular (EST-SSRs) markers-based extents of genetic diversity analysis as well as chromosome number and ploidy level determination using 174 accessions from diverse agro-ecologies in Ethiopia. For agro-morphological diversity analysis, the experimental samples were tested at Ambo and Holeta agricultural research centres, using an alpha lattice design at the locations and three blocks/replication followed by appropriate management practices. Agronomic and morphological traits-based data were collected on twenty-eight (12 qualitative and 16 quantitative) traits at the right growth stage and analysed using SAS v9.0, MINITAB® v14.13 and FigTree v1.4.3. Cytogenetic characterization was also carried out using very young root tips generated from soil covered stem rings, followed by appropriate pre-treatment, fixation and maceration. For molecular genetic diversity analysis, genomic DNA was extracted form silcagel dried young leaves collected from 287 plants (1-3 plants per accessions) following CTAB protocol. EST-SSRs marker were designed from Plectranthus barbatus cDNA sequences deposited in the GenBank, followed by PCR amplification, capillary electrophoresis, peak identification, and scoring. The scored allele size data were analysed for polymorphism, diversity indices and genetic relationship and structure using windows compatible applications. The agro-morphological traits considered showed varied morphotypes in all of the leaf (four in leaf color, three in leaf arrangement, three in leaf shape), stem (three in stem color, two in each of stem spot and stem spot colors) and tuber (four in tuber skin color, three in each of tuber texture, tuber shape and tuber hair) characterstics. Similarly, the traits revealed a wide range of variability in mean performance (minimum range of 1.39 – 2.13 cm observed in tuber diameter and maximum range of 112.90 – 165.10 xvii days observed in days to 50% flower initiation) and variance components among and within the accessions. Similarly, the mean square of all the traits showed a highly significant (P< 0.001) variation among the tested accessions. Such wide variation suggests the presence of variability which can be exploited through selection. Several of the traits showed a slightly greater or nearly equal phenotypic coefficient of variation (PCV) to that of genotypic coefficients of variation (GCV), suggesting larger contribution of the genotypic effect for phenotypic expression of such characters and hence, phenotypic values-based selection for the traits may be effective. High estimates of GCV (>23%) coupled with high estimate of heritability (Hb%) (>94%) and high genetic advance as a percent of mean (GAM) (>46) were revealed in tuber weight per hill, number of primary branches per plant, number of tubers per hill, and number of plants per hill indicating the importance of such traits for selection in Ethiopian potato improvement programs. The significantly positive phenotypic, and genotypic correlation in tuber weight per hill and number of tubers per hill with each other and several other traits as well as their negative phenotypic correlation with some other traits indicates the direction of selection. Moreover, the significantly higher absolute magnitudes in genotypic correlation compared to their corresponding phenotypic correlation suggest the genetic base of those traits. Cytogenetic characterization revealed a very smaller sized metaphase chromosome with a count of 2n = c.56 and hepta- or octa-ploidy was speculated on the bases of basic chromosome number reports (x = 8 or 7) for the species of genus Plectranthus and other members of the Lamiaceae family. Such chromosome count and ploidy level report could serve as a baseline information in selection and crosshybridization of Ethiopian potato with other closely related species. In total, twenty new polymorphic expressed sequence tag based simple sequence repeat (EST-SSRs) markers have successfully been developed and used in genetic diversity analyses. The marker detected a total of 128 alleles (6.4 alleles per locus) over the entire loci and populations with effective number of alleles ranging from 1.06 - 3.17 (an average of 1.67). The marker showed an overall highest (94.17%) percent polymorphism, and extents of PIC in the range of slightly informative to highly informative suggesting the potential of those developed markers as a valuable genetic tool and resource to evaluate the extent of genetic diversity and population structure of not only Ethiopian potato but also various other species within the Lamiaceae family. The ranges and levels of mean observed heterozygosity (0.33 – 0.429), Shanon’s information index (0.523 – 0.663), and Nei’s gene diversity (0.307 – 0.384) across loci showed a medium degree of variation among the populations which is a direct reflection of sharing of most of the alleles among the populations that partly resulted from high overall gene flow (Nm=18.29). Comparatively, Wenbera (Wen), Wolaita Sodo (WSo), Hadiya and Kambata-Tembaro (HKT) and Southwest Shewa (SwSh) populations could be considered as Ethiopian potato diversification and in-situ conservation sites. xviii Hierarchical analysis of molecular variance (AMOVA) showed significant but low population differentiation with at most 3% of the total variation in each of the groupings, such as among the populations, among geographic regions, and among regions of accessions. Likewise, cluster analysis in all the cases and STRUCTURE analyses did not group the populations into sharply distinct clusters, which could be attributed to historical and contemporary gene flow and/or the reproductive biology of the crop. In conclusion, this study has wider implications in bringing such a ‘super-neglected’ crop to the scientific agenda and thus, opens up the door to its improvement and conservation. However, it is important to exhaust all areas and regions in the country and more number of SSRs or other up-to-date molecular marker systems to come up with more accurate level of genetic diversity estimates.
Alkaline Protease of Alkaliphiles Isolated from Ethiopian Rift Valley Soda- Lakes
(Addis Ababa University, 2000-05) Hasana Azaga; Abate Dawit (PhD); Gesesse Amare (PhD)
A prntcolytic alkali phi lie bacteria was selected out of the bacterial isolates obtained from Riftvallcy soda lakes. The isolate was obtained from lake Chitu water sample. The organism was Gram variable aerobic, rod shaped spore forming, motile bacterium. It has been identified to belongs to the genus Bacillus. The growth of the isolate coded as CH-W 1 was observed in the pH and temperature ranges between 7-l l and 25-40°C respectively. Protease production was observed shortly afler inoculation reaching to maximum after 48 h. The crude enzyme had a temperature optimum of 55 °C and a pH optimum of 9. The enzyme was stable in a broad pH range of 8.5- l 0.5 after l hr incubation at 50 ° C . It has a half-life of 30 min. at 60°C . The enzyme was slightly activated by CuSO4 . Where as Ba++ ,Zn ++, Co++ , Hg++ , Mn ++, Fc+r ,.Na', K'and Ca" had very little.or no effect cin the activity of the enzyme. The enzyme was strongly inhibited by I mM PMSF sh o\,,ing that it belongs to the class of serine protease. EDTA at a concentration of IO mM partially inhibits the activity. This shows the requirement of Ca'' for stability. The organism was efficient in degrading Nug meal (GuizotaAbyssinia) and feather when used as a sole carbon source.
Amebiasis in Ethiopia: Problems in diagnosis and determination of prevalence of infection
(Addis Ababa University, 2005-02) Kebede Amha; Petros Beyene (PhD); Polderman Anthon (PhD)
Although in Ethiopia intestinal amebiasis is believed to be associated with many cases of diarrhea, diagnosis is based on examination of fresh stool samples by microscopy, a method that cannot discriminate the potential invasive Entamoeba histolytica from the commensal Entamoeba dispar. The annual reports from Wonji Hospital and the Ethio-Netherlands AIDS Research Project indicate intestinal amebiasis to be a common infection with the highest prevalence compared to other parasites. Despite a considerably high reporting of intestinal amebiasis, a twenty-year hospital records among 117080 admitted patients showed only 47 suspected liver abscess cases, suggesting overdiagnosis. Though the prevalence of E. histolytica/E. dispar by microscopy was 24.9 % in Wonji and Akaki, the specific PCR did not confirm the presence of any E. histolytica infection. Even after careful microscopic analysis, by using quality control measures on 246 patients with diarrhea, microscopy demonstrated 40 % positivity of Entamoeba infection. However, application of PCR, a molecular diagnostic method that can distinguish E. histolytica from E. dispar did not confirm any E. histolytica, only 9% harboured E.dispar. Coproantigen detection ELISA in the same patients showed clear lack of sensitivity and specificity whereby only 11.4 % specimens in the genus Entamoeba specific ELISA were in agreement with the PCR, and none of the eight E. histolytica antigen positive was confirmed. The absence of E. histolytica infection in Wonji and Akaki was better justified with lower seropositivity (3 %) finding, using recombinant surface antigen of E. histolytica. Further study among healthy primary-school students and prisoners emphasize the high occurrence of E. dispar infection. Each of these samples was checked for Entamoeba infection, by careful microscopy with ocular measurement, of formol–ether concentrates. DNA was then extracted from the 213 samples (27.6%) found Entamoeba-positive, and run in a real-time PCR with primers, based on the SSU-rRNA gene sequences of E. histolytica and E. dispar, that allow DNA from the two species to be distinguished. Although E. dispar DNA was identified in 195 (91.5%), no E. histolytica DNA was detected. This finding is consistent with the previous investigation that many amebic infections in Ethiopia are incorrectly attributed to E. histolytica and then treated unnecessarly. In order to further confirm the actual occurrence of E. histolytica, 110 suspected haematophagous trophozoites were collected from different hospitals and health centers. Only three (2.7 %) E. histolytica cases were detected by real-time PCR, while 71.3 % were E. dispar. The finding was similar among HIV/AIDS patients with diarrhea where microscopy revealed 12 % E. histolytica/E. dispar, but none with E. histolytica. The tradition of microscopy in a routine diagnostic set-up appears unsatisfactory to reliably differentiate RBCengulfing ameba from non-invasive ameba. The public health implication of this study is that patient management and validity of epidemiological surveys are questionable as long as microscopy is the only diagnostic tool. There is also a need to continue work to establish the aetiology of diarrhea wrongly associated with amebae and explain the enigma of patients recovering following “anti-amebic” treatment. The commonly reported complaints of bloody mucoid diarrhea and association of low CD4 with E. dispar infection among AIDS patients require alternative explanation. Training in microscopy needs improvement, if not to diagnose the infection accurately, at least to minimize the over-reporting. This work emphasizes the necessity of capacity building for important diarrheal pathogens with molecular diagnostics at referral level
Amylase Production by two Microbial Isolates: Isolation, Enzyme characterization, and optimization of Cultivation condition
(Addis Ababa Universty, 2017-03) Leykun, Senaite; Gessesse, Amare (PhD)
Amylases are commercially important enzymes commonly used for the hydrolysis of starch to generate hydrolysates for different food and nonfood applications. Although Ethiopia has a huge potential for the production of starch, to date the starch industry is nonexistent and the country import large quantities of starch hydrolysates from abroad. The aim of this study was production of industrially important microbial amylases through solid state fermentation (SSF), optimize the cultivation condition, and characterize the enzymes. Screening using starch containing media one bacterial isolate producing α-amylase, designated as BI151, and one fungal isolate producing glucoamylase, designated as FI97, were isolated from soil. Both isolates grew well on SSF with wheat bran as a solid substrate. BI151 α-amylase produced 4,636 U/g of amylase after 96 h of incubation at 50% moisture. Isolate FI97 produced a maximum activity of 4,674 U/g after 72 h of incubation under SSF at a moisture content of 67%. For both isolates supplementation with organic or inorganic nitrogen source did not have any impact on enzyme production. This suggests that wheat bran supplemented with mineral salts and water has enough nutrients to support growth of the organisms and make the production process a lot cheaper. Amylase BI151was optimally active in the temperature range of 65-75°C with a peak at 70°C and in the PH range of 4.5 -5.5. On the other hand amylase of FI97 was optimally active at 65°C and at pH 6. These properties showed that both enzymes have properties that make them potentially suitable for starch hydrolysis, especially in the brewery industries. Under lab scale evaluation, both enzymes were able to efficiently hydrolyze high concentration of starch adjuncts (60% of the malt adjunct mixture) indicating their potential for application as malt enzyme supplements. Key word: α-amylase, Glucoamylase, starch hydrolysate, relative activity, residual activity
Amylases of Potential Industrial Application from Microbial Sources
(Addis Ababa Universty, 2006-07) Teka, Muluye; Gessesse, Amare (PhD)
Glucoamylase and alpha amylase producing fungal and bacterial strains were studied. The fungal isolate previously isolated by Abreham Tesfaye belongs to the genus Aspergillus and was designated as Aspergillus No 43 (Asp 43). The bacterial isolate (designated as Bacillus sp CRC) which was isolated from Hot spring around lake Chitu was Gram positive, rode ,motile catalase positive and central endospor former. Asp 43 glucoamylase was optimally active at pH 4 and temperature of 65 OC. where as Bacillus CRC alpha amylase showed maximum activity at a pH range of 5- 6 and temperature of 80 OC. Addition of 5 mM Ca2+ did not affect the temperature profile of both types of amylases. However the bacterial amylase was Ca dependent for its themostability, and this enzyme retained about 53 % of its original activity after 3 hr incubation at 80 OC. In addition Bacillus CRC was stable over a broad pH range retaining more than 80 % of its original activity in the pH range of 4.5 – 8.0 . Asp.43 grown in SSF medium at 25 OC produced maximum enzyme when the moisture content of the wheat bran used as a sole carbon source was 66.7 %. The optimum pH and temperature for maximum enzyme production by Bacillus CRC in liquid medium was 6.0 and 55 OC respectively. Bacillus CRC produced maximum enzyme when the sole carbon and nitrogen sources were starch and trypton ( at 0.5 and 0.2 % respectively). Enzyme secretion by the fungal and bacterial isolates reached maximum after 96 and 36 hr of incubation respectively.
Analyses of Phenotypic and Molecular Diversity, Genotype by Environment Interaction and Food-Feed Traits in Tef [Eragrostistef (Zucc.) Trotter]
(Addis Ababa University, 2018-12-05) Jifar, Habte; Tesfaye, Kassahun (PhD); Dagne, Kifle (PhD)
Tef [Eragrostis tef (Zucc.) Trotter] is an indigenous major cereal crop widely grown and utilized in Ethiopia. It is an excellent source of healthy and nutritious human food and livestock feed. However, its productivity is significantly lower than that of major cereals grown in Ethiopia. Although diverse constraints contribute for its inferior grain yield, the huge diversity present in tef germplasm has not been exploited. This study was, therefore, conducted to assess the phenotypic and molecular diversity, genotype by environment interactions and variation of food-feed traits in tef genotypes from various sources. The field experiments were conducted at Holetta, Debre Zeit, Alem Tena, Adet, Axum and Shambu using randomized complete block design with three replications whereas, the laboratory analyses were conducted at the University of Bern in Switzerland, Sci-Co in South Africa, International Livestock Research Institute (ILRI) in Ethiopia and International Crops Research Institute for Semi-Arid Tropics (ICRISAT) in India. Phenotypic diversity was assessed based on 12 quantitative and five qualitative traits of 188 tef genotypes while, the genotyping study was conducted using 189 genotypes with 16 SSR markers. Results from phenotypic study revealed the existence of huge variability in plant height (60.7 to 107.1 cm), panicle length (19.5 to 39.5 cm), number of fertile tillers per plant (2.1 to 5.5), number of spikelets per panicle (156.7 to 441.7), thousand kernel weight (20.7 to 33.0 mg), grain yield (3.7 to 7.3 t/ha) and lodging index (44.7 to 79.3%). Molecular diversity study generated a total of 260 alleles for the total studied loci. Cluster and STRUCTURE analyses showed four gene pools, but with high level of admixtures due to geographic proximity and/or trans human seed exchange. Analysis of molecular variance showed significant variation of 56.6% within individuals, 40.8% among individuals within population and 2.6% among populations. Based on SSR study, furthermore, a core collection composed of 64 genotypes (33.9%) is suffice to capture the entire alleles of studied genotypes. Significant variations were also detected for all traits due to genotypes, environments and their interaction. Based on AMMI, GGE and Eberhart Russell’s model, G6 (Melko) was identified as the highest yielding and moderately stable variety while, E5 (Debre Zeit-1) was the highest yielding and ideal environment. GGE biplot grouped the test environments into four mega environments and the studied genotypes into seven groups. Analysis of food-feed traits revealed significant (P<0.01) effect of genotypes, year, locations and their interactions. Based on this, the highest mean crude protein (CP), in vitro organic matter digestibility (IVOMD), metabolic energy (ME), total biomass (TBM) and straw yield (STY), and the lowest mean for neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were obtained from genotypes evaluated at Debre Zeit. Cluster analysis and PCA-biplot grouped the genotypes into four. These are, (i) those with improved feed quality traits such as Kena, Genet and Ambo Toke;(ii)) those with high food-feed traits such as Dukem, Gimbichu and Dega Tef; (iii) those with dual purpose such as Melko, Koye and Key Tena; and (iv) those with low food-feed traits such as Simada, Etsub and Mechare. The overall results of the present study enabled to identify tef genotypes with sufficient morpho-agronomic and molecular variations, better yield and stability, and food-feed traits having implications for collection, conservation and proper utilization of germplasm which are the basis of tef improvement.
Analyses of Phenotypic and Molecular Diversity, Genotype by Environment Interaction and Food-Feed Traits in Tef [Eragrostistef (Zucc.) Trotter]
(Addis Ababa University, 2018-12-02) Jifar, Habte; Dagne, Kifle (PhD); Tesfaye, Kassahun (PhD)
Tef [Eragrostis tef (Zucc.) Trotter] is an indigenous major cereal crop widely grown and utilized in Ethiopia. It is an excellent source of healthy and nutritious human food and livestock feed. However, its productivity is significantly lower than that of major cereals grown in Ethiopia. Although diverse constraints contribute for its inferior grain yield, the huge diversity present in tef germplasm has not been exploited. This study was, therefore, conducted to assess the phenotypic and molecular diversity, genotype by environment interactions and variation of food-feed traits in tef genotypes from various sources. The field experiments were conducted at Holetta, Debre Zeit, Alem Tena, Adet, Axum and Shambu using randomized complete block design with three replications whereas, the laboratory analyses were conducted at the University of Bern in Switzerland, Sci-Co in South Africa, International Livestock Research Institute (ILRI) in Ethiopia and International Crops Research Institute for Semi-Arid Tropics (ICRISAT) in India. Phenotypic diversity was assessed based on 12 quantitative and five qualitative traits of 188 tef genotypes while, the genotyping study was conducted using 189 genotypes with 16 SSR markers. Results from phenotypic study revealed the existence of huge variability in plant height (60.7 to 107.1 cm), panicle length (19.5 to 39.5 cm), number of fertile tillers per plant (2.1 to 5.5), number of spikelets per panicle (156.7 to 441.7), thousand kernel weight (20.7 to 33.0 mg), grain yield (3.7 to 7.3 t/ha) and lodging index (44.7 to 79.3%). Molecular diversity study generated a total of 260 alleles for the total studied loci. Cluster and STRUCTURE analyses showed four gene pools, but with high level of admixtures due xix to geographic proximity and/or trans human seed exchange. Analysis of molecular variance showed significant variation of 56.6% within individuals, 40.8% among individuals within population and 2.6% among populations. Based on SSR study, furthermore, a core collection composed of 64 genotypes (33.9%) is suffice to capture the entire alleles of studied genotypes. Significant variations were also detected for all traits due to genotypes, environments and their interaction. Based on AMMI, GGE and Eberhart Russell’s model, G6 (Melko) was identified as the highest yielding and moderately stable variety while, E5 (Debre Zeit-1) was the highest yielding and ideal environment. GGE biplot grouped the test environments into four mega environments and the studied genotypes into seven groups. Analysis of food-feed traits revealed significant (P<0.01) effect of genotypes, year, locations and their interactions. Based on this, the highest mean crude protein (CP), in vitro organic matter digestibility (IVOMD), metabolic energy (ME), total biomass (TBM) and straw yield (STY), and the lowest mean for neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were obtained from genotypes evaluated at Debre Zeit. Cluster analysis and PCA-biplot grouped the genotypes into four. These are, (i) those with improved feed quality traits such as Kena, Genet and Ambo Toke;(ii)) those with high food-feed traits such as Dukem, Gimbichu and Dega Tef; (iii) those with dual purpose such as Melko, Koye and Key Tena; and (iv) those with low food-feed traits such as Simada, Etsub and Mechare. The overall results of the present study enabled to identify tef genotypes with sufficient morpho-agronomic and molecular variations, better yield and stability, and food-feed traits having implications for collection, conservation and proper utilization of germplasm which are the basis of tef improvement.
Analysis of Five Year District Health Office Data for Intestinal Parasitosis in Aleta-Chuko Sidama Ethiopia
(Addis Ababa University, 2021-09-15) Degife, Teshale; Mamo, Hassen (PhD)
Intestinal parasitic infections (IPIs) are widely distributed, particularly in low-income countries. The objective of this study was to retrospectively assess the prevalence of IPIs among preschool (1-4 year) and school-age (5-18 year) children in Aleta-Chuko District (ACD), Sidama Region, South Ethiopia, in the past five-year (September 2015 - August 2019). The study was retrospective study. It used secondary data from disease registers of ACD health office (2015 - 2019) to assess the prevalence of IPIs among preschool and school-age children.Overall, 199,241 children visited district health centers during this period and 140,706 (65,292 males, 75,414 females) were school-age children. Of these, 50,057(25.1%) were positive for IPIs with slightly more females (13.2%) than males (11.9%). The number of children diagnosed for IPIs had increased continuously although there was some decline in 2016 and 2017. Six different types of intestinal parasites were identified. Ascaris lumbricoides was the most prevalent (41.5%) followed by Entamoeba histolytica/dispar/moshkovskii (14.3%). The least identified parasite was Schistosoma mansoni. Intestinal parasites remain major health problem among preschool and school-aged children in ACD and emphasis on control measures including periodic deworming is needed. Comprehensive community-based studies are required for better understanding of the impact of IPIs on children in ACD.
Analysis of Hwuoral Immune Response to a Panel of P!aslllodilllllja!cipallllll Blood-Stage Vaccine Candidate Antigens in Nanmtlly Primed Populations in Seasonal Malaria Settings in Ethiopia
(Addis Ababa University, 2012-06) Mamo Hassen; Petros Beyene (Professor)
In Ethiopia, the general population is quite vulnerable to unpredictable cyclic epidemics of Plasmodium falciparum malaria. However, there is very little information on the anti-malaria immune profile of the population residing in the endemic regions of the country. This study was designed to investigate the nature of humoral immune response to malaria in two population groups in two endemic localities, Shewa Robit in the north and Boditi in south. In a cross-sectional study, the study participants were diagnosed for malaria infection microscopically and by the rapid diagnostic test (RDT). The sera were tested by using enzyme-linked immunosorbent assay (ELISA) for total immunoglobulin (Ig) G against P. falciparum blood-stage vaccine candidate antigens: apical membrane antigen 1 (AMA1), glutamate-rich protein (GLURP) R2 region, and merozoite surface protein 2 (MSP2) allelic variants (3D7 and FC27) in Shewa Robit. Total IgG against GLURP-R0, MSP3 and GMZ2 and IgG subclasses against GLURP-R0 and MSP3 were assayed in both Shewa Robit and Boditi sera. Whereas 23(8.6%) blood-smear-positive cases for P. falciparum were detected in Boditi, all Shewa Robit study participants had no detectable P. falciparum infection. At both localities total IgG prevalence and levels to GMZ2 were significantly higher than the response to the component domains (GLURP-R0 and MSP3) indicating the induction of strong GMZ2-specific natural antibodies. There was significant difference between the median antibody level to GMZ2, GLURP-R0 and MSP3 compared to the responses to other antigens tested in Shewa Robit, indicating that GMZ2 could be a more relevant blood-stage malaria vaccine candidate antigen. Higher total IgG and subclass prevalence and levels were detected in Shewa Robit than Boditi, suggesting difference in the intensity of malaria transmission in the two localities and/or genetic differences between the two population groups in their response to blood-stage P. falciparum antigens. In both study sites, IgG subclass antibody levels to GLURP-R0 were significantly higher than that to MSP3 for all corresponding subclasses in most individuals, indicating the higher relative immunogenicity and protective potential of GLURP-R0 compared to MSP3. Against both GLURP-R0 and MSP3, the ratio of cytophilic to noncytophilic antibodies was >1 in the majority of the study participants, in both study sites, indicating the induction of protective antibodies against the two antigens. Analysis of age-related pattern in antibody levels against the antigens tested showed a positive association with increasing age for most antigens suggesting the role of intrinsic agerelated factors in immune maturation. The age factor appears plausible as there was no evidence for increase in antibody response with increasing frequency of reported past clinical malaria. Overall, the study has shown that Ethiopian population groups residing in unstable and seasonal malaria epidemiological settings have a high prevalence and levels of long-lived antibodies that readily recognize P. falciparum blood-stage vaccine candidate antigens, particularly GMZ2 and its component fractions (GLURP-R0 and MSP3). Furthermore, detection of high level antibody responses in non-febrile smear-negative individuals without history of reported past malaria episodes may possibly be an indication of a low-grade, asymptomatic (submicroscopic) infections in the induction and maintenance of high level protective immunity. Therefore, to determine the implication of submicroscopic infections in the induction and boosting of malaria immunity versus the existence of long-lived malaria-specific antibodies in the absence of boosting from submicroscopic infection, PCR confirmation of the microscopy-negative samples would be necessary. Keywords: antigen, blood-stage vaccine, cytophilic IgG subclass, ELISA, Ethiopia, falciparum malaria, noncytophilic IgG subclass
Analysis of T Cell Subsets in HIV-L Infected and un Infected Ethiopians on the Basis of Various Differentiation and Activation Markers in the Context of Intestinal Parasitic Infections
(Addis Ababa Universty, 2001-06) Kassu Afework; Petros Beyene (PhD)
The present study assessed whether expansion of a specific sub-population of CD8+ T cells is responsible for variations in CD8+ T cell counts; and also investigated the effect of incident intestinal parasitic infection and treatment on the profile of various differentiation and activation markers on CD4+ and CD8+ T cells from HIV-1 infected and uninfected adult Ethiopians (n=150). The pattern of CD8+ T cell population in whole blood samples of 86 subjects (60 HIV negative and 26 HIY positive) from Akaki and Wonji were analysed. The effect of incident intestinal parasites and their treatment was studied on cryo preserved PBMCs of 64 subjects (41 HIY- and 23 HIV+). In both cases, the samples were stained with antibodies to various T cell differentiation and activation markers and naiVe, memory, effector, memory/effector, activated and resting CD4+ and CD8+ T cell subsets were quantified by triple colour FACScan. Significantly higher (P
Anti inflammatory and bacterial effect of Arthrospira plathensis (Spirulina) on Schistosomiasis mansoni and Selected Human Bacterial Pathogens
(Addis Ababa University, 2011-02) Sileshi, Abraham; Lakew, Mekuria (PhD)
Arthroscopic platen sis (spirulina) is multi cellular filamentous blue green alga that is abundantly found in lakes of high salinity in Ethiopia. Several studies have shown that it has importance in human health and nutrition. The health benefits of spirulina relies mainly on tits anti-inflammatory, anti- oxidant and antimicrobial properties. In the current study spirulina wasstudied for its anti-inflammatory and antimicrobial effects using change in size and number of S.mansoni induced granular in infected mice and inhibitions of known human bacterialpathogens (S. typhi, S. dysenteriae, E. coli, S. aureus, S. epidermidis, C. freundii, and P.aeruginosa) as parameters. S. mansoni is a parasitic helmintic infection with high public healthimportance next to malaria, HIV-AIDS and TB especially in sub Saharan countries. Chronicinflammatory response in response to S. mansoni egg trapped in liver tissue is the mainpathology responsible for the morbidity and mortality associated with the disease.Methodologies used in the study involve S. mansoni infection of Swiss albino mice, treatment ofexperimental animals with spirulina at two doses 100 and 400 mg/kg through oral, rectal, andoral/rectal. Animal dissection and histological staining of the liver to measure granulomadiameter, and finally antibiotic sensitivity test using disk diffusion method was done.Independent T. test comparison of the measurements form the anti-inflammatory test showed that the size of granular around an egg from spiritual treated compared to controls was significantly reduced. In association with this, comparison between different treated groups showed that treatment through oral route at a dose of 400 mg/ kg scores the highest percentage reduction both in granular size (59.53%) and granular number (72%). Result from the antimicrobial study showed that all test organisms except S. typhi were susceptible for the water suspension and extracts of spiritual unlike that of the Methanol extract. The possible explanations are discussed. In conclusion and recommendation the anti-inflammatory and antimicrobial effect of spiritual are summarized and future direction of research indicated Key words: - Spirulina, S. mansoni, Granuloma, Anti-inflammatory, Antimicrobia
Antibacterial Activity of Moringa Stenopetala against Some Human Pathogenic Bacterial Strains
(Addis Ababa University, 2012-06) Chekesa Basha; Mekonnen Yalemtsehay (Professor)
An emerging of antibiotic resistance brings most serious public health problems. It is therefore, important to look for more effective, safer and less toxic alternate options of treatment. The aim of the present study was to investigate antibacterial activity of Moringa stenopetala against some human pathogenic bacteria using disk diffusion method and agar dilution for minimum inhibitory concentration. The result revealed that, most of the plant extracts had antibacterial activity. Staphylococcus aureus was found to be the most susceptible bacteria to crude 80% methanol extract of seeds and ethyl acetate extract of root barks with inhibition zones of 18.66±0.88mm and 16.00±1.15mm and minimum inhibitory concentration of 1.25mg/ml and 2.5mg/ml respectively, whereas Pseudomonas aeruginosa was the most resistant bacteria to all of crude extracts. Similarly, Staphylococcus aureus was the most susceptible bacterial strain to chloroform fraction with inhibition diameter of 28.00±0.57mm and minimum inhibitory concentration of 0.31mg/ml, while Pseudomonas aeruginosa was the most resistant strain with inhibition zone of 9.66±0.33mm and minimum inhibitory concentration of10mg/ml respectively. In conclusion, this study is not only proves antibacterial activity of Moringa stenopetala, also provides a scientific basis for their traditional use. Pure chemical compounds and antimicrobial activity against many fungi and bacteria should be studied to use them as sources and templates for synthesis of drugs to control infectious diseases. Key words and phrases: Antibacterial activity, Moringa stenopetala, traditional medicine, extracts, fractionation, MIC, solvents
The Antibacterial, Atioxidant and Toxicity Studies of Crude Aerial Part Extracts of Satureja Punctata (Benth.) Briq.
(Addis Ababa University, 2015-08-05) Melaku Mesfin; Mekonnen Yalemtsehay (Professor)
The ever-increasing demand for antimicrobial products against resistant infectious (microbial) agents necessitates their search from microorganisms and medicinal plants. The Satureja punctata (locally known as “lomishet”, Amharic) is one of such plants used as herbal medicine in different parts of Ethiopia. The objective of this study is to evaluate the antimicrobial and antioxidant activities of the crude aerial part extract of the plant against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa and its toxicity on animal mouse model. The aerial parts were collected from Entoto and Sendafa and extracted using absolute ethanol, water and n-hexane. The Antibacterial activity was assessed using disc diffusion method, and the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the extracts were determined by the agar dilution method. The radical scavenging activity of the extracts was evaluated by DPPH assay. The result indicated that the solvent extracts exhibited antibacterial activities on the test organisms, except the n-hexane and the activities were dependent on solvent type and concentration. Consequently, the maximum antibacterial activity was displayed by the aqueous extract against S. aureus(10.33±0.25mm), E. coli (9.83±0.19mm) and P.aeruginosa (8.53±0.12mm) and by ethanol extract against S. aureus (10.33±0.41mm), E. coli(9.37±0.18mm) and P.aeruginosa (10.17±0.46mm) at the concentration of 600mg/ml. Furthermore, the MIC values for both ethanol and water extracts were from 75 to 100 mg/ml; whereas the MBC values for ethanol and water extracts were from 100 to 150mg/ml and100mg/ml, respectively indicating that the ethanol-extract was slightly more effective than the water extracts. The in vitro free radical scavenging activity showed that the aqueous, ethanol and n-hexane extracts showed maximum antioxidant activity of 87.89%, 78.68% and 47.18% at 25mg/ml concentration, respectively. Thus, the plant showed interesting antioxidant activity that could be useful in the management of oxidative stress. Oral administration of crude ethanol extract at the dose of 2000 mg/kg did not show any mortalities or evidence of adverse effects on the Swiss albino mice, implying that S. punctata is non-toxic to the animal model.
Antimicrobial Activity of Lactic Acid Bacteria Isolated From ‘Ergo’, Ethiopian Traditional Fermented Milk, on Some Foodborne Pathogens
(Addis Ababa University, 2006-11-04) Assefa, Esayas; Beyene, Fekadu
“Ergo” is a traditional Ethiopian fermented milk produced by spontaneous fermentation using traditional utensils under non-hygienic environment. To produce microbiologically safe and chemically defined product; study on the production, processing, utilization and handling of ‘Ergo’ under different agro-ecological zones as well as the isolation and characterization of potential lactic acid bacteria strains with the potential production of inhibitory factor should be the first essential step. In this study ninety-smallholder farmers from the three districts (Lumme, Fentale, Adami Tulu), thirty from each district, who produced and processed milk were purposeively selected and individually interviewed using a semi-structured questionnaire to asses the production, processing, utilization and handling of traditional fermented milk, “Ergo”. Milk fermentation takes relatively longer period (3.57 + 1.04 days) in Lumme district than the two districts Fentale and Adami Tulu (1.03+ 0.183). All households (100%) in Fentale consume un-boiled milk; rather they utilize it in its fermented or raw state, where as only 40% and 46.6% of households in Lumme and Adami Tulu areas, respectively, used fermented milk for consumption. Even though milk is fermented and consumed in all these areas a very limited hygienic care is practiced, 93.4% of the households do not heed for the sanitary aspect of the milk and milk products. To evaluate the antimicrobial activity of the lactic acid bacteria (LAB), 112 strains of LAB, belonging to Lactococcus, Lactobacillus, Entorococcus, Streptococcus, Leuconostoc and Pediococcus were isolated from Ethiopian traditional fermented milk, “Ergo”. Moreover the culture filtrates of all the isolates were examined for antimicrobial activity on some food born pathogens Salmonella thyphi, Shigella flexineri , Staphylococcus aures (ATCC-25923) and Escherchia coli (ATCC-25922) using disc diffusion assay method. Inhibition diameters obtained with the inhibitory substance of 2mm and above inhibition zone over the control were considered as positive. Twelve strains of lactic acid bacteria that produced the greatest antimicrobial substance were selected. These strains were identified as species of Lactobacillus acidophilus(2) , Lactobacillus plantarum(2), Lactococcus lactic spp crimoris(2) , Lactococcus lactic spp lactic(3), Leuconostoc lactic (1) , Pediococcus pentosaceus(1) and Pediococcus sp.(1). All the inhibitory substance-producing strains were tested for their temperature and pH stability. The antimicrobial activity of the culture filtrate of isolated strains were completely inactivated when treated at 121oC for 15 minutes, where as at 30, 60 and 80oC there were no Significant (P>0.05) difference in diameter of inhibition compared to untreated (control). The entire culture filtrate were stable with in a wide range of pH (2-10) and no significant (P>0.05) difference was observed between the treated filtrate and the untreated (control). However treating the culture filtrate at pH 12 has significantly (P<0.05) decreased the inhibition diameter.
Arbuscular Mycorrhizal Fungal diversity of Coffee and Shade trees of Bonga forest, South Western Ethiopia.
(Addis Ababa Universty, 2006-07) Chanie Tadesse; Assefa Fassil (PhD)
To date, information regarding densities and identification of dominant coffee shade trees and explanation of their interaction with physical parameters in southwestern part of Ethiopia is extremely very scanty. Moreover, mycorrhizal fungi spore densities, colonization rate, diversity and interaction with physical and chemical parameters of soil are not evaluated in any of the coffee forest in the country and this work is to bridge such information gap. So, reconnaissance survey was conducted and the number of coffee and coffee shade tree species was counted with in a 20m x 20 m quadrat. Spores were separated from the bulk of the soil by the wet sieving and decanting method followed by sucrose centrifugation and counted per 100 g dry soil. Percent root colonization was determined clearing the root segments in 10 % KOH and stained with 0.05 % trypan blue.During the reconnaissance survey extremely large number of coffee seedlings and saplings were observed in the study area. Olea capensis, Schefflera abyssinica, Phoenix reclinata and Prunus africana were encountered as dominant shade tree species with percentage occurrences of 15, 12, 11 and 10 %, respectively. Milletia ferruginea, Albizia gummifera, Cordia africana, Croton macrostachyus and Dracaena steudneri comprise 40 % of the total number of plant species. Twenty four percent of the dominant shade trees were principally represented by leguminous trees such as M. ferruginea and A. gummifera. Arbuscular mycorrhizal fungal status was also investigated. Investigation of roots and rhizosphere soils of 14 shade tree species, M. ferruginea, S. abyssinica, C. macrostachyus, Ficus vasta, A. gummifera, O. capensis, C. africana, Ehretia abyssinica, Pouteria adolfi-friederici, Pavetta oliveriana, Ficus sur, P. africana, P. reclinata and Polyscias fulva, and the coffee tree underneath revealed arbuscular mycorrhizal colonization and different types of AM spores. The number and morphological type of spores were determined in 100g of rhizosphere soil. All species had a high level of AM colonization. F. vasta, C. macrostachyus, S. abyssinica and O. capensis spp. had the highest level of colonization. E. abyssinica, P. africana and P. fulva exhibited the lowest level of colonization. The coffee trees revealed generally maximum colonization than respected shade trees. The number of spores forming AM in all rhizosphere soils ranged between 578-1313 spores/ 100g of dry soil. Spores were grouped in four principal morphological types Glomus, Gigaspora, Scuttelospora and Acaulospora in descending order of relative abundance, respectively. Spore density and colonization percentage were found to be negatively correlated with O.C, total N, available P, EC and Zn. Population of spore types were also some how determined by soil physical and chemical properties. Arbuscular Mycorrhizal fungi are commonly considered to be non specific with respect to their host and their ability to colonize particular plant species. Conversely, it is investigated that plant species differ in their ability to form and benefit from mycorrhizal relationship. This study didn’t investigate the whole range of organisms in the rhizosphere which could give benefits to trees. So, it is difficult to draw conclusion that the well being of the shade trees and coffee plants in the natural forest is due to only AMF colonization. However, AMF could be considered to be a future tool in agriculture, i.e. fertilizer substitute and as biocontrol agent. So, it is recommended that: (a) studying the ecology, diversity and host range of AMF is of enormous importance before application. (b) their relation with nutrient dynamics and other soil characteristics should be evaluated before use. (c) these organisms should receive the proper management. (d) the impact of litter fall on AMF spore density and diversity should be evaluated in the future. Finally, this symbiosis command great interest as the subject of future research with relation to food security

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