Microbial, Cellular and Molecular Biology

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Identification of Serotypes and Assessment of' Multiple Drug Resistance In 360 Shigella Isolates
(Addis Ababa University, 1980-06) G/Yohannes, Afework
Three hundred and sixty urban and rural Shigellae isolates were analysed in respect to serogrouping, serotyping, biochemotyping and drug resistance. S. flexneri (50.55%) was most common, followed by £. dysenteriae (32.78%), ~. b~£ii (11.39%) and £. sonnei (5.28%). Isolation rates of £. ftysenteriae, £. flexneri and fl. sonnei were eomparablo in rural and urban areas, except for fl. boydii which is more common in urban areas (significant at P=0.05). Out of the thirty-two known Shigella serotypes, twenty-two were identified in this study (i.e. fl. dysenteria~ 1,2, 3, 4, 6, 7; ,2. flexneri I, 2, 3, 4, 6; E.. boydii 1, 2, 3, 4, 5, 8, 9, 10, 12, 111; and E.. sonnei). Urban isolates were represented by 22 serotypes compared to only 11 in rural areas. E.. dysenteriae serotypo 1 (Shiga's bacillus) Was more common in rural areas (34.48%) than in urban areas (17.22%), and this difference is· significant (P=O,05). Ninteen patterns of drug resistance were observed, with TSu (21.11%), TCACbSSu (19.72%) and TSSu (12.50%) being comparatively more common. There \'Iere nine patterns of drug resistance in 8erogroup A, 12 patterns in ll, 8 patterns in C, and 5 patterns in D. TCACbSSu pattern (53.39%) in serogroup A, TSu (41.76%) in B, and TSSu (26.31%) in D Vlere observed. lIitnin serogroup A, 81.82% of fl' dysenteriae serotype 1 was associated with the TCACbSSu pattern. There were Ii patterns of resistance in urban areas compared to 10 in rural areas. The prominence of TCACbSSu pattern in rural areas was related to the Iligh isolation rate of fl. dysenteriae type 1 in these places. In rural areas, 14.94% of Shigella isolates were classified as sensitive to all drugs tested, compared to 26.37% in urban areas and this difference is significant (P=0.05). Identification of rare biochemotypes included: a) three strains of mannitol negative ~. flexneri type 6, b) a strain of gas productng ~. boydii type 14, nnd c) a strain of 'invasive' ~. ££1i (0:164) serologically cross reacting in ~. dysenteriae serotype 3· antiserum. Based on the present study, it is recommended that: a) a further study of Shigella be persued, b) a Shigella reference center be established, c) an improvement of sanitation be stressed, d) chemotherapy be discourae;ed and e) a national policy for drug sale be enacted.
In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology (Applied Genetics)
(Addis Ababa University, 1990) Gebremariam Zelalem; Bekele Endashaw (Professor); Tesfaye Kassahun (PhD)
Three Lepus species are currently considered to occur in Ethiopia: Lepus habessinicus Hemprich and Ehrenberg 1832 (Abyssinian Hare), L. fagani Thomas 1903 (Ethiopian Hare) and L. starcki Petter 1963 (Ethiopian Highland Hare) based on limited morphologic and cytogenetic data. Analyses of morphological characters, 13 microsatellite loci, one mitochondrial (ATPase sub-unit 6 sequence) and five nuclear sequences (Transferrin (TF), Thyroglobulin (TG), Uncoupling protein two (UCP2), Uncoupling protein four (UCP4) and Thyroid stimulating hormone beta (TSHB)) were made to provide an initial comprehensive data on the evolutionary relationship among the three hare species collected from twenty six localities in Ethiopia and identified by their external phenotypes, dental and skull characters. In addition, other Lepus species: L. capensis, L. saxatilis, L. europaeus, L. timidus and L. victoriae were used for phylogenetic comparison. Multivariate skull morphometric analyses (PCA & DA) of seven Lepus species (n = 327) indicated clear distinction among the species. Lepus habessinicus had a distinct morpho shape space relative to L. capensis. Lepus fagani had a distinct morpho shape space relative to L. saxatilis and L. victoriae. Lepus starcki had a distinct morpho shape space relative to L. europaeus and L. capensis. Microsatellite analysis based on hares (n = 107) collected from Ethiopia resulted in clusters of hare species in accordance with population origin and collection sites. Individuals of L. habessinicus and L. fagani were substructured in accordance with population of origin and collection localities with some degree of SSR introgression among each other. Microsatellite introgressions were documented also between species. In all analyses (structure, FCA and NJ) L. starcki individuals were consistently clustered into a single group. All phylogenetic reconstruction methods used (Neighbour Joining, Maximum Likelihood, Bayesian Analysis and Median Joining Networks) by and large resulted in similar topologies for mitochondrial and nuclear sequences, respectively. The three hare species from Ethiopia were not monophyletic for both mitochondrial and nuclear sequences, but showed signs of both unidirectional and bidirectional introgressions among each other. Lepus habessinicus carried mtDNA distinct from South and North African L. capensis, with no sign of introgression, contrary to earlier suggestions to include L. habessinicus under L. capensis. Similarly, L. starcki had distinct mtDNA relative to L. capensis and L. europaeus as opposed to previous suggestions to include L. starcki either under L. capensis or L. europaeus. Despite the morphological distinctness of L. fagani, it shared a substantial proportion of mtDNA and nucDNA with hare species from Ethiopia and L. saxatilis from South Africa. The nuclear sequences revealed phylogenetically and geographically meaningful clusters, albeit with a fair amount of indications of recent or historical introgresions among the species. In line with ATP, the nuclear sequences indicated close phylogenetic relationships among the three Lepus species from Ethiopia, with L. fagani being surprisingly tightly connected to L. habessinicus. Furthermore, the microsatellite analysis indicated low levels of genetic differentiation (G’ST and Dest 0.384 and 0.334, respectively) between L. habessinicus and L. fagani. Moreover, the nuclear sequences suggested a closer evolutionary relationship of Cape hare from north and south Africa to hare species in Ethiopia. Although the nuclear sequence analyses supported the North American ancestry of Lepus, the results of the present analysis also suggested Africa as a potential ancestral area of Lepus. Overall, both multivariate skull morphometric and molecular analyses suggested a specific status of L. habessinicus, L. fagani and L. starcki. Keywords: Ethiopia, introgression, Lepus habessinicus, Lepus fagani, Lepus starcki, microsatellite, mtDNA, nucDNA, phylogenetics, phylogroup and skull morphometry.
Morphological and Biochemical Diversity of Emmer Wheat (Triticum Dicoccum (Schrank) Schub.) in Ethiopia
(Addis Ababa University, 1993) Tesfaye Kassahun; Bekele Endashaw (Professor); Asfaw Zemede (PhD)
MOIphological and isozyme diversity study of 55 emmer wheat (TriticuIII dicocculII (Schrank) Schub.) populations and the associated ethnobotanical knowledge in Arsi and Bale have been undertaken in this study. A total of 55 landrace populations (25-45 single plant per population which is a total of 1885 entries) were considered for mOlphological study. Protein content was determined for the 37 emmer wheat populations following the standard procedure. The dl)' malter, ash, organic matter and Ca were done for 55 emmer wheat population following the standard protocol. The isozyme variations in the three enzyme systems: esterase (EST), acid phosphatase (ACP) and aspartate aminotransferase (AA T) were investigated for 12 emmer wheat populations selected after clustering them on mOlphological characters. Mean, coefficient of variations, heritability, genetic advance, correlation coefficients cluster analysis, chi- square test, multidimensional scaling, and discriminant analysis were done for each mOlphological character. Correspondence analysis and Shannon's diversity index (H,) were used to estimate the morpholOgical variation. The coefficient of variation was large for most traits in Shewa indicating more diversity in this region. This was further confirmed by Shannon's diversity index where the mean diversity (H ') was the highest in this region (0.78) followed by Harerghe and Gonder, the least diversity index was shown by Tigray region. H' at population level ranged frOIll O(lIIonomOlphic) to 1 (poIYIIIOlphic) for sOllie traits. The overall mean diversity for Ethiopia was estimated to be H' ~ 0.75. Farlllers in the study area assert/hat emmer wheat is mOlphologically uniform and this was confirmed through field observation. This crop has important traits in disease resistance, stress tolerance and traditional use values. Fanners in Arsi and Bale regions have their own folklore, songs, and sayings on this particular crop to indicate its use value and problems related with its hulledness. Phenotypic polymO/phism was observed for the three ellZymes. Relatively higher variation was observed for esterase ellZyme i>ystem and no variation was detected for enzyme system aspartate amino transferase (AAT). Positive (though not significant) correlation was observed between mOlphological diversity index and mean heterozygosity based on isozyme data. The Arsi and Bale area niakes an important in-situ conservation site for em/ner wheat because of the specialty of the crop to the society. Key words: Triticum dicoccum, emmer wheat diversity, morphological character, protein, isozyme, ethnobotany
Isolation and Characterization of the 34 Kd Gene of Mycobacterium Leprae.
(Addis Ababa University, 1993-09) Abebe, Feseha; W.M., Peter (PhD)
An M. Iprae::pHC79 cosmid genomic library was screened for the identification, isolation and characterization of the 34 kd antigen gene of Mycobacterium leprae using its homologue, the 34 kd antigen gene of Mycobacterium tuberculosis, as a DNA probe. Qut of a total of 79 different cosmids screened, covering about 88 % of the genome of M. leprae, one cosmid designated as BL 1527 was found to harbor the gene of interest. Restriction enzyme analysis of the cosmid BL 1527 yielded four DNA fragments - two containing the complete gene and two containing part of the gene. The four DNA segments were subcloned in plasmid vectors pUCBM21 and pBKS+ and characterized physically and immunologically. Physical characterization of the gene by nucleotide sequencing revealed that the 34 kd antigen gene has an open reading frame of 801 nucleotides encoding 266 amino acid residues bearing a molecular weight of28,887 daltons and an isoelectric point of 4.3. Homology comparison with the 34 kd antigen gene from M.tuberculosis has revealed 80.6 % (633 nucleotides) identity at the DNA level and 85 % (221 amino acids) identity at the protein level. Immunological characterization by western blot analysis has shown that the gene is expressed in E. coli from its own promotor signals and that the recombinant protein is recognized by the monoclonal antibody F-126. 2 which is specific for the mycobacterial 34 kd antigens. Crossed Immuno-electrophoresis analysis has established that the recombinant 34 kd antigen corresponds to the non secreted Antigen 84 of mycobacteria.
Response of Ethiopian Hiv -1 to Azidothymidine Dideoxyinos Ine and Interferon-Alpha
(Addis Ababa University, 1993-09) Wondimu, Zenebech; Strannegtlrd, Orjan(Professor); Forsgren, Marianne (PhD)
Human Immunodeficiency Virus (HIV), the aeti%gic agent of Acquired Inullllllodeficiency Syndrome (AIDS) is known to manifest high degree of genetic variability. Based on the genomic structure two types of HIV, HIV-l and HIV-2 are known to date. The presence of a distinct strain of HIV-l in Ethiopia has been reported. Differences in the pattern of viraemia and immunologic reactions from the one documented in European and North American patients has been observed in Ethiopians infected with this virus. Treatment with azidothymidine (AIT) has been shown to result in decreased serum levels of P2-microglobulin (PrM), tumour necrosis factor alpha (TNF-a) and neopterin in the European and North American HIV-l patients. Whether there are differences in sensitivity to antiviral compounds, such as AZf, dideoxyinosine (ddl) or interferon alpha (IFN-(J.) in Ethiopian HIV-l strains and the effect of treatment on the immunological reactions were not known. The in vitro sensitivity of 8 Ethiopian strains to antiviral compounds was investigated in parallel with 8 Swedish strains all from untreated AIDS patients. The antiviral effects were measured by p24 antigen capture EUSA and Reverse transcriptase activity assay. A pilot controlled study of 200mg and 600mg oral AZT was conducted in 53 HIV-l infected Ethiopians. The treatment effects were evaluated by assessment of the clinical status of the patients and the measurement of serum levels of PrM, TNF-(J. and neopterin. No statistically significant difference in drug sensitivity was found between the Ethiopian and The Swedish strains to All, ddl and IEN-(J. in vitro. In the majority of the treated patients improvement in their clinical conditions was reported and a gain in body weight was observed. A statistically significant decrease in the levels of PrM, TNF-(J. and neopterin were found in the treated patients in contrast to the control patients.
Investigation on the Genus Bacillus in the Control of Mosquitoes
(Addis Ababa University, 1993-10) Wahab Abdel; Abate Dawit (PhD); Gemetchu Teferi (PhD)
Screening programmer to isolate strains of the genus Bacillus for their potential use in the control of mosquitoes was done. Over 90 spore-forming bacillus strains were examined. Six isolates AA-2, AA-6, AA-10, AA-l1, AA-71, and AA-82) have sho\m killing ability of which four (AA-2, AA-11, AA-71, and AA-82) were found to be effective. Biochemical and morphological characterizations have revealed similarity between these isolates and the Well Known mosquito pathogens (B. thuringiensis ancl B. sphaericus). The effect of temperature and aeralion supply on biomass production was studied. Isolateswere found to dliffen:nlly responcl to temperJIUle. Isol"Ias fnunu to qro\•: h'l'll at 30')C. A,;-ll 11ds been found to huve a widerange of temperature (23- 40)AA-71 has Sl10WIl io\'erse relation \,ith temperature, h'hereas isolate "\A-82 demonstrated critical decrease in growth with increase in temperature and 30~ was found to be optimal. Dry biomass was founcl to increase with the increase in aeration levels. The efficacy of the isolates was tested on different larval stages of mosquito. LC IO that ranged from 43-170 llg/ml \,as obtained. ~o siqnificant difference has been observed in sensitivity of different larval stages of mosquito to different bacteria at a significance level of 1% except isolate AA-11. Efficacy of the isolates under natural conditions was also tested. Some strains (eg. AA-82) have shown good results.Further confirmatory characterization is needed as well as investigation for optimal conditions to increase toxin yield.
A Study on Human Antibody Response to Moraxella (Branhamella) Catarrhalls Antigens During Respiratory Tract Infections
(Addis Ababa Universty, 1996-05) Gebreselassie, Solomon; Hathaway, Audrey E. (PhD)
In this study, M. catarrhalis was isolated from 68 of 200 (34.0%) sputum samples of patients with community-acquired pneumonia. It was also found in 56 (28.0%) of the nasopharynx of these patients. There were 42 (61.8%) males. and 26 (38.2%) females with pneumonia caused by M. catarrhalis. Fifty one of the 68 (75.0%) had chronic underlying diseases. Of the isolates from sputum 37 (54.4%) and 32 (57.1%) of the 56 nasopharyngeal isolates were p-Iactamase positive. An ELISA was used to determine the antibody response of sera of patients with pneumonia caused by M. catarrhalis to the outer membrane proteins (OMPs) of the bacterium for both IgG-A-M and IgG31eveis in the sera. It was found that 40 of the 68 (58.8%) patients with pneumonia caused by this organism showed significant rise in the total immunoglobulin (IgG-A-M) level, and 43 of 68 (63.2%) in the IgG3 level in their convalescent sera. In the control sera only I of 30 (3.3%) patients had significant rise for IgG-A-M while none of them showed significant increase in titre for IgG3 (P< 0.05). Using lipopolysaccharide (LPS) antigens, only 16 of 68 (23.5%) paired sera showed significant increase in antibody titre for both IgG-A-M and IgG3 compared to 2 of 30 (6.7%) of controls (P< 0.05). Significant bactericidal activity of the convalescent-phase sera at a dilution of 60% was detected in l3 of 15 (86.7%) of the sera assayed. The same sera without complement were used as controls and none of the controls showed significant bactericidal activities (P< 0.05).
Diarrhoea Associated Parasitic infectious Agents In Patient S With Aids in Selected Addis Ababa Hospitals
(Addis Ababa University, 1996-05) Fisseha Bitew; Petros Beyene (PhD)
The study was aimed at determining the prevalence of gastrointestinal parasites in AIDS patients with chronic diarrhoea. This prevalence was compared with that in two control groups: HIV seronegative diarrhoeal patients and AIDS patients (HIV seropositive individuals) without diarrhoea. Stool specimens were screened for parasite infection from clinically diagnosed hospitalized AIDS patients in seven hospitals in Addis Ababa. Of 147 AIDS patients with chronic diarrhoea, 74 (50.3%) were infected with one or more kind of parasites. Out of the 56 non-AIDS (seronegative) diarrhoeal patients 23 (41.1%) and out of the 43 non-diarrhoeal (seropositive) patients 18 (41.9%) were infected by a variety of intestinal protozoa and helminths. The parasites detected in AIDS patients only were crptosporidium spp, Isospora spp, and Blastocystis spp, Ascaris lumbricoides, Giardia lamblia, strongyloides stercoralis, Taenia saginata, Trichuris trichura, Entamoeba histolytica, and Hook worm spp. Among the intestinal parasites, Cryptosporidium spp was exclusively associated w.i th diarrhoeal AIDS patients (P < 0.001). None of the other parasites were significantly associated with AIDS patients. The high proportion of the study subjects who had diarrhoea in the absence of identifiable parasitic infections suggests that other infectious agents (eg. Bacteria and Virus) or mechanisms other than infectious agents are responsible for the diarrhoea.
The Prevalence and Growth Potential of Some Salmonella Species in "Kitfo" and Effect of "Kitfo" Preparation Processes on the Test Organisms
(Addis Ababa University, 1997-05) Tegegne Mezegbu; Ashenafi Mogessie (PhD)
In an attempt to assess the microbial load and the prevalence of salmonellae in "kitfo", a total of 50 raw or "Tire kitfo" samples were collected from ten food establishments (hotels, restaurant and bars) from different areas of Addis Ababa. The microbial load in "tire kitfo" samples collected from all sources was markedly high. The highest average loads of aerobic mesophilic bacteria (8.33 Log(CFUlg», coliforms (6.77 Log(CFUlg», staphylococci (7.29 Log(CFUlg», lactic acid bacteria (5.37 Log(CFU/g», molds and yeasts (5.96 Log(CFUlg», and aerobic spores (5.67 Log(CFUlg» were encountered in "kitfo" from three hotels and one bar. On the other hand, the lowest average loads of aerobic mesophilic bacteria (7.30 Log(CFU/g», coliforms (4.51 Log(CFU/g», staphylococci (6.18 Log(CFU/g», lactic acid bacteria (4.10 Log(CFU/g», molds and yeasts (4.06 Log(CFUlg», and aerobic spores (3.71 Log(CFU/g» were found from one restaurant and two hotels. Variation within samples in counts of coliforms, staphylococci, lactic acid bacteria, molds and yeasts, and aerobic spores was significant in all or most establishments (CV> 10%). However, counts of staphylococci and lactic acid bacteria did not show marked variations among hotels, restaurant and bars. Variation within samples in the count of aerobic mesophilic bacteria in most food establishments was not significant (CV < 10%). The microbial load of slightly cooked "kitfo" ("Iebleb kitfo") was found to be high, though it was less than that of raw or "tire kitfo". Variation in the counts of aerobic mesophilic bacteria, staphylococci and lactic acid bacteria in "lebleb kitfo" atllJHg ten establisluIleIlts was liul slgiiincalit (CV ~ 10%), while thaI of coliforms, molds and yeasts, and aerobic spores was significant (CV> 10%). No microorganisms have been detected from "tibs kitfo", except very few aerobic spores. From the study of the prevalence of Salmonella in "kitfo" twenty one positive samples were obtained from the 50 samples collected from the above hotels, restaurant and bars. Among these positive samples of Salmonella, the highest number of positive ';)«IHpJ"''' • ..I 1HUIl!'1'-''' \"J.o II)) n"'l1. .... llVlll .... "'1,,11 VI -l.A.Vl ..... l.L1 UIiU .LIUl ..", UIlU Lll\'; JVW .... .,l HUIllU1.,.J Vi positive samples (one) was from Hotel G. No Salmonella was isolated from "kitfo" of Hotel D, E and Restaurant A. No Salmonella was encountered in "lebleb" and "tibs kitfo" samples. Of the three selective enrichment broths, i.e. Selenite Broth, Thetrathionate Broth, and Rappaport- Vassiliadis Medium used for enriching the growth of Salmonella species, RVB was found to be superior. Of the four plating media, i.e., Salmonella-Shigella (SS)agar, MacConkey agar, Brilliant Green agar (BG) and Xylose Lysine Desoxycholate (XLD) medium, BG was found out to be superior in supporting the growth of salmonellae encountered in this work. The microbial load of swabs from raw beef was relatively low when compared to that of "kitfo". The loads of aerobic mesophilic bacteria, coliforms, staphylococci, lactic acid bacteria, molds & yeasts and aerobic spores were found to be 5.07 Log(CFU/cm'), 2.34 Log(CFU/cm'), 3.14 Log(CFU/cm'), 2.63 Log(CFU/cm'), 2.54 Log(CFUlcm'), and 2.60 Log(CFU/cm'), respectively. When assessing the growth potential of Salmonella species in "kitfo", they were found to grow weI!. They grew to the level of 7.0 Log(CFU/g) within 12h of incubation.
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.
Heiminthes & Immune Responses to Mycobacterial Antigens
(Addis Ababa University, 1999-05) Elias, Daniel; Britton, Sven (Professor)
Tuberculosis has become the major killer disease globally. As it is the case with most other infectious diseases the magnitude of the problem is the highest in resource poor countries. There are several means to control the disease. One of these is the use of vaccine (BCG). However, the vaccine offers the least protection against puhnonary tuberculosis, the most infectious fonn of the disease, in areas where TB is most prevalent. Part of the explanation for this may be that chronic infectious disease particularly, hehninthes, in these areas may polmize the immune response towards humoral type when cell mediated inununity takes the lions share in defending the body against intracellular infections. In order to assess whether intestinal helminthes could influence mycobacteria specific immunity, we investigated the ill vivo and ill vitro mycobactelia specific immune responses of helminth exposed population at Kotebe Teacher's Training College. Our finding was that dewonning significantly enhanced the ill vitro lymphocyte proliferative responses, IFN-y and IL-IO production in response to mycobacterial antigens and a T cell mitogen, PHA, compared to untreated controls. Moreover, the initially PPD non reactive subjects were BCG vaccinated and later tested for mycobacteria specific immune responses ill vivo and ill vitro. We found that BCG, when given after deworming caused significant improvement in T cell proliferation and IFN-y production compared to pre-vaccination levels in response to a mycobacterial antigen, PPD, whereas in the placebo group the difference was not significant. This finding support the notion that intestinal hehninthes may impair immune responses against mycobactelial infections and that BCG vaccination may confer better protection if given after dewonning in helminth exposed population which is an already established notion in veterinary medicine.
Morphological and Biocuemical Diversity of Grassplj/L (Lathylws Sativus L) in Etiilol'la
(Addis Ababa University, 1999-05) Tadesse Wuletaw; Bekele Endashaw (Professor)
Fifty grasspea populations that were selected based on administrative regions and different altitude classes were used in tins study. A total of 1199 entries were evaluated for 14 qualitative and quantitative morphological traits. Oxalyl diamino propanoic acid (ODAP) variation was determined for 150 entries by taking three entries fi'Om each population. Protein content was detenrriued for the 50 grasspea populations following a standard procedure. TIle phenotypic variations in three enzyme systems: esterase(EST EC 3.1.1.), acid phosphatase (ACP:EC 3.1.3.2), and aspmiate amino tranferase(AAT:EC 2.6.1.1) was investigated using horizontal starch gel electrophoresis for 10 populations (25 entries per population) selected from the cluster analysis result based on the morphological data. Mean, coefficient of variations, heritability, genetic advance, cOlTeiation coefficients, cluster analysis, factor analysis and Shannon's diversity index estimate were used to estimate the morphological variation. ANOVA (analysis of variance) result indicated that there was significant variation for the different traits witlrin a population and between populations. The coefficient of variation was large for most traits in Gonder indicating more diversity in this region. Tins was fmiher asceliained by Shannon's diversity index where the mean diversity (H' ) was the highest in this region (0.65) followed by Tigray region (H' = 0.64). H at population level ranged form 0 (monomorphic) to 0.9 (polymorphic) for some traits. The over all mean diversity for Ethiopia was estimated to be H' =0.61. Chi-square analysis also showed that there was highly significant variation for each traits (seed size, flower colour, leaflet size and pattem of testa). Some variation in altitude groups was also noted though it was not significant. Analysis of variance for mean ODAP content and protein content showed non- significant differences between populations. However, ODAP analysis from single plants showed significant variation both with in a population and between populations. Fortunately, four individual plants with low ODAP contents ranging from 0.14% to 0.18% (range with in safe level) were obtained from populations collected from different regions indicating the diversity and variation of this trait with in population and between regions. These low ODAP lines are obviously considered important for further breeding program. Phenotypic polymOlphism was obselved for the three enzymes. High phenotypic polymOlphism was observed for esterase. There was negative bnt non significant (t= -0.25) association between morphological diversity index and mean expected hetrozygosity base on isozyme data. Key words:Lthyrus sativa, grasspea, lathyrism, diversity, morphological characters, ODAP, protein, isozyme, conselvation.
Morphological and Molecular Diversity in Durum Wheat (Triticum DurumDesf.) Landraces of North Shewa
(Addis Ababa University, 1999-05) Tamiru Mulualem; Asfaw Zemede (PhD)
Morphological and molecular diversity study and the associated ethnobotanical profile of durum wheat landraces (farmers' varieties) of North Shewa have been undertaken in this study. Morphological diversity stndy on thirteen durnm wheat popnlations (381 genotypes) ,collected earlier by the Biodiversity Conservation and Research Institute from five awrajas and ten weredas of North Shewa region, was conducted using six heritable qualitative traits. The diversity was estimated using Shannon Weaver diversity index (H'). H' was estimated for each population, wereda, awraja and altitudinal class and the entire collection. On population basis H' is lowest at Basona Warena wereda (0.25 ± 0.08) and highest at Kimbibit Wereda (0.65 ± 0.16). On awraja basis H' ranged from (0.50 ± 0.03) in Gam Midir to (0.73 ± 0.10) in Selale. On altitudinal basis, the third altitudinal class (2401-2600 m.a.s.l.) sc ored the highest H' (0.78). The diversity index for the study area is very high (0.79 ± 0.08). On character basis seed color showed the highest H' (0.97). Only glume pubescence was conspicuously unique to specific awmja and altitudinal class. A high diversity of the crop is also observed from the ethnobotany study. A total of twenty-five different farmers' varieties having different vernacular names were recorded. The environmental, social (cultural) and economic factors were identified by the farmer as factors for maintaining the existing diversity. The different varieties have important merits in agronomy, disease resistance, traditional use values and so on. Farmers have their own folk taxonomy system for identifying and classifying their varieties. The Randomly Amplified Polymorphic DNA (RAPD) molecular technique with a gluten-coding single primer system with the following sequence, { 5'- ATG AAG ACC TTC CTC GTC TT- 3'} { 5'-AAC GCC GAA TGG CAC ACT A- 3' } showed good demonstration of the inter-population diversity of the thirteen populations. Except three populations, all the rest showed diffel'ent banding patterns with different base pair fmgments for the same primer used. Generally the whole study confirmed the existence of strikingly high diversity of durum wheat in the study area and underlines the role of the indigenous knowledge of farmers for the existing diversity. Key words: Wheat, Triticum dUrlIm, landraces (fanners' varieties), diversity index (H') Morphological traits, ethnobotany, RAPD.
Some Microbiological and Biochemical Studies on the Fermentation of two Traditional Condiments,Awaze'and'Datta
(Addis Ababa University, 1999-06) Idris, Ahmed; Ashenafi, Mogessie (Professor)
The microbial dynamics, pH and titratable acidity together with some biochemical changes associated with the fermentation of two Ethiopian condiments, 'awaze' and 'daffa' were investigated following the traditional methods of preparation. The two condiments were prepared in the laboratmy, from a variety of vegetable and spice n:ixtures. The fermentation of both condiments is carbohydrate fermentation carried out by lactic acid bacteria with a progressive increase in titratable acidity and decline in pH. Other microorganisms isolated did not appear to playa major role in the process of fermentation, although yeasts appeared in the latter stage of 'awaze' fermentation. Lactic acid bacteria, which kept on increasing throughout the fermentation, ranged from 106 -lOll cfu/g for 'awaze' and from 104-109 f9r 'datta '. Enterobacteriacea and coliforms, which were isolated at the initial stages of 'datta' fermentation, were inhibited at 32 h. by the antimicrobial activity of the dominating lactic acid bacteria. Reducing sugar changes, detected by the DNScolorimetric method, was very significant in 'awaze' than 'datta " in which there was an initial increase, and a decline afterwards due to the utilization of sugar by the proliferating lactics. The changes associated with the protein content of the two condiments, as detected by the formol titration method, were very little.
Microbiological Profile and Safety of Some Street vended Foods in Addis Ababa
(Addis Ababa University, 1999-06) Muleta, Diriba; Ashenafi, Mogessie (Professor)
Microbiological profiles and safety of street-vended food collected from vendors in Addis Ababa, Ethiopia were determined in 1998 and 1999. The 150 samples of foods investigated consisted of sambllssa (legume-based), cooked and sauced macaroni, lentil sandwich, kitfo (raw minced and spiced meat dish) and egg sandwich. All samples of kitfo and egg sandwich were held at temperatures that permit bacterial growth (15.5-24S C) and (15.5- 34S C), respectively. Most samples of the other food products were stored at temperature range of 15.5-34S C. In general, sambllssa had low counts of aerobic mesophilic bacteria and staphylococci. Aerobic bacterial spores, yeasts, members of Enterobacteriaceae, colifonns and Bacillus cerellS were isolated rarely in sambllssa. Highest aerobic mesophilic counts (> 108 cfb/g) were recorded in egg sandwich. The other food products contained counts as high as 107 cfu/g. Presumptive Bacillus cereus attained greatest population level in macaroni samples (> 10' cfu/g). In other dishes it had counts> 104 cfuig. Majority of the samples which were positive tor aerobic bacterial spores, coliforms, Enterobacteriaceae, staphylococci and yeasts contained >104 , 10', 105 , 105 , and 104 cfuig, respectively. In the street foods considered in this study, moulds were either detected rarely or not at all. Variations in counts among the samples of the respective street foods were obvious (CV 10%).A total of 1552 bacterial isolates were screened from foods obtained from street vendors and were characterized into various genera and bacterial groups. Gram-positive organisms predominated (76.9%) along with Staphylococcus and Micrococcus species comprising 22.8% and 15% of aerobic microflora, respectively. From Gram-negative organisms, members of a family Enterobacteriaceae (14.5%) were frequently encountered. Salmonella was recovered from samples of egg sandwich and kitfo and macaroni yielded Shigella. Challenge studies showed that the test strains, Salmonella typhimul'illlll, Shigella flexneri and Staphylococcus aureus grew in egg sandwich, kitfo and macaroni to markedly high levels. The faulty handling and serving operations, poor sanitation of the vending sites, time-temperature abuse during holding, addition of raw products either to cooked or uncooked dishes, the use of unclean multipurpose cloth and water of dubious quality (reused water) were deemed potentially hazardous. Thus, education of both vendors and consumers in basic principles of hygiene is an urgent issue by concerned bodies.
The Relative Importance of Three Cabbage Aphid Species (Brevicoryne Brassicae, Lipaphis Erysimi; Myzus Persicae) and Their Parasitoid Complex in Kenya.
(Addis Ababa, 2000) Gacii, Peter; Seyoum, Emiru(PhD)
Studies on the relative importance of the three known species of cabbage aphids (Brevicoryne brassicae (Linneaus), Myzus persicae (Sulz) and Upaphis erysimi (Kaltenbach)) and their parasitoid complex in Kenya were carried out. The studies involved farmers' fields' surveys and interviews and also field experiments conducted at the Jomo Kenyatta University of Agriculture and technology (JKUAT) Research and Production Department land and in a farmer's field at Limuru. Gloria F1 hybrid was sown at JKUAT while Copenhagen Market variety was sown at Limuru. The field experiments were conducted in a Completely Randomized Block Design (CRBD) involving three treatments and a control, with three repl icates each. The treatments comprised of applications of: - Lambda-Cyhalothrin (Karate; Bacillus thuringiensis Berliner (Bt.) var. Kurstaki toxin (Dipel 2x) that selectively kills Diamondback moth (DBM); A combination of the two pesticides (Karate and Bt.) and Untreated controls. Parameters included; relat ive abundance of the three species of aphids; their corresponding damage to cabbage; the number of mummies per sample; percent parasitism and the proportions of the primary- and hyper-parasitoids hatched. The parasitoid complex was constructed from the identified parasitoids. The results showed aphid counts to be higher in non karate-treated plots than in the karate-treated ones. More hyperparasitoids notably Alloxysta sp and Pachyneuron sp. than the only primary parasitoid (Diaeratiella rapae) emerged from non karate-treated plots. This might suggest that karate might have the advantage of repelling hyperparasitoids or inhibiting their emergence. Yield showed some negative correlation with aphid infestations. A combined treatment gave the best overall yield at both sites.
Secies Composition of Sulfur Oxidizing and Denitrifying Bacteria from the Tannery Wastewater Treatment Plant, Modjo, Ethiopia
(Addis Ababa Universty, 2000-03) Bekele Sissay; Assefa Fassil (PhD)
The discharge of tannery effluents to different water bodies increase pollution because of heavy accumulation of toxic compounds such as sulfur and nitrogen containing organic wastes, chromium, arsenic etc. Thitty-six strains of Sulfur Oxidizing Bacteria (SOB) belonging to sixteen genera were isolated fi'om the three tannery ponds of a Tannery Waste Treatment system at Modjo. They were characterized on the basis of their cultural, morphological, and physiological characters. The genera were Acidiphilium, Acromatium, Macromonas, Sulfolobus, Thermothrix, Thiobacilllls, Thiobacterium, Thiomicrospira, Thiosphaera, Thiospira, Thiovullim Rhodobacfor, Rhodocyclus, Rhodomicrobillm, Rhodopseudomonas, and Rhodosprillllm. Out of these isolates, twenty-one strains falling into twelve genera were capable of denitrification. Bacterial enumeration based on MPN technique showed a population number between 1.2 x 103 and 1.1 x 106 . The number of strains recovered from the different ponds varies such that twenty, sixteen, and nine strains were isolated fi'om AFP, SFP, and MP, respectively. The dominant strains isolated from different pond systems were the genera Thiobacilllls (S strains), Rhodopselldomonas, (4 strains), and Rhodomicrobiulll (3 strains). The most frequently isolated strains in the three different pond systems were Thiobacilllls (AUS9) and Rhodolllicl'obilllll (AU142). Isolated strains showed variation in growth on different media and some such as Acidiphiliulll (AUS); Slilfoloblis (AUl2); Thiobacillus (AUS9) Thiolllicrospira (AU62); Thiosphaera (AUS7) Rhodolllicl'obilllll (AUl42, AU164, AUl76) failed to grow on denitrifYing medium but were capable of growing on nitrogen free (nit) medium. Most of the isolates were found to be slow growers reaching to their exponential stage between 4S-72 hours. In their spatial distribution, the anaerobic strains in AFP system and the aerobic isolates in the SFP and MP systems were found to be dominant.
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.
In vitro T-Cell Responses as Surrogate Markers for HIV -lInfection Progression in Ethiopia
(Addis Ababa University, 2000-06) Legesse, Mengistu; Lakew, Mekuria (PhD)
In an effort to use immunological abnormalities as markers of HI V -I infection progression in Ethiopian individuals, this work assessed the changes in in vitro and in vivo T-cell responses to recall M. tuberculosis antigen, purified protein derivative (PPO). This was further evaluated against C04+ and C08+ T-cells count and viral load in the same subjects. Peripheral blood mononuclear cells (PBMCs) isolated from 36 HIV -I seropositive and 105 seronegative individuals were subjected to 6 days in vitro PPO stimulation and cytokine production. The T-cell proliferative response was then evaluated using 5-bromo-2-deoxyuridine (BrdU) instead of thymidine incorporation as a method of assay. Cytokines (IFN-y and IL-4) production was measured in the PBMC culture supernatants using sandwich ELISA. Three days phytoheamagglutinin (PHA) stimulation responses were used as positive controls. In vivo tuberculin PPO responses were measured for 18 HIV -I seropositive and 74 seronegative individuals by standard Mantoux reaction. Results show that T-cell responses to PPO both in vitro and in vivo were affected by HIV -I infection, in contrast to HIV -I seronegatives. Significant reduction in IFN-y production was also observed between the two groups following PPO stimulation (p = 0.000), but not with IL-4. Thus, a shift from Thl to Th2 cytokines production was not observed. Analysis of T --;;ell responses to PPO showed a positive correlation with C04+ T cell counts, and negative with plasma viral load. This confirmed that HIV -I infection progression is followed by a depletion of C04+ T cells count and high viral load. Change in proliferation and cytokine production was specific to PPO. It suggests that Alycobacteriulll tuberculosis specific immune responses are affected in HIV -I infected sUbjects. Taken together our results indicated that evaluating T-cell responses to recall antigen, PPO can be used as an early marker for HIV -I infection progression.of unemployment, sexual promiscuity and the presence of a wide diversity of HIV-I subtypes in Africa have also been indicated as powerful components in HIV -I spread and transmission. The virus has profound economic and social implications for both developed and developing countries. No country in the world is free of the disease and can claim that it has stopped its spread. The most effective and efficient method to prevent HIV -infection and disease manifestation would be through vaccination and effective treatment, which currently are not available. The generally accepted method of implementing preventive strategies for HIV infection is education that will inform and increase awareness of the society
Variablit Some Microbiol Ogical and Chemical Properties of Tej Between and Within Some Vending Houses in Addis Ababa
(Addis Ababa University, 2000-07) Bahiru, Bekele; Ashenafi, Mogessie (Professor)
'tej' being honey wine, is one of the primitive types of wines that are not crystal clear, but cloudy, effervescent, containing residues of substrates, the fermenting yeasts and other microorganisms. Honey wines are prepared and consumed in many parts of the world. 'tej' is consumed widely in many parts of Ethiopia and is prepared from honey, or honey and sugar, water and Gesho (Rhamnus prenoides). Roots, stems or leaves of plants are added assuming atlincrease in the alcohol content and its potency. Like any other spontaneous fermentation, 'tej', in its fermentation, relies on the microorganisms that inhabit the must from different sources. The substrates, utensils, equipment, and the envirorunent are responsible for the randomization of the micro flora. In such spontaneous fermentation and un-optimal physical and chemical conditions, variability in the product quality and stability becomes inevitable. So, investigating the variability and its extent in some microbiological and chemical properties of 'tej' at the time it was ready for consumption, was the objective of this study. At consumption stage, 'tej' was highly loaded with yeasts and Lactic acid bacteria. Members of different genera of yeasts, members of the bacterial genera of Lactobacillus, Pediococcus, Streptococcus, Leuconostoc, were isolated and identified. Of the yeast isolates, species of Saccharomyces were dominant, followed by Kluyveromyces, Debal'O/11yces, Hansel1ula, Endomycopsis and Pichia specIes. Generally, the homofermentative (52.09%) were higher in counts than the heterofennentative( 47.91 %) lactic acid bacterial population.. All the isolates from the aerobic mesophilic bacterial plates were identified as members of Baci I/lls. All the studied microbiological and chemical properties of 'te}' had significant variability among samples within each vending house. Also, except yeast and lactic acid bacterial populations and fusel oil contents, all the other microbiological and chemical properties of 'te} , studied had statistically significant variability (AN OVA, p < 0.004 for all) between the mean values of all the vending houses. Generally, all samples also showed variability in their total alcohol, total lipid, protein, and reducing sugar. Variability in fusel oil, titratable acidity and total carbohydrate was also noted. So, 'te}' at the time it was ready for consumption had statistically significant variability both within and between vending houses in the microbiological and chemical properties.

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