Academic submissions (CoVAB)

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Assaying for thermostable DNA polymerase activity in putative theromophilic bacteria isolates from selected garbage dumping sites and hot springs, in Uganda

(Makerere University, 2018) Tusuubira, Sharifu

Molecular and biotechnological research and training in Uganda is hampered by the high cost of acquisition of the required/appropriate chemicals and reagents, notably the DNA polymerase enzyme. Subsequently, there is insufficient practical training in molecular techniques, especially the Polymerase Chain Reaction (PCR) based, at tertiary institutions. This study set out to explore bacterial thermophiles from selected compost pits and hot springs in Uganda for production of thermostable polymerases as an alternative source for use in practical training and biotechnology. The study assessed the basic bacteria cell morphology and colony characteristics; and utilized genotypic methods of 16sRNA sequencing together with Naїve Bayesian Classification to offer a statistical test to flag taxa differentially represented between samples. Putative novel bacteria were isolated from selected garbage compost sites and geothermal hot springs in Uganda. The genotyping revealed that an isolate from compost UG-Wampewo belonged to Proteobacterium at 60% degree of confidence; while the hot spring isolates, Ug Kiga_Mud2,and Ug Kiga, are probably a novel species under Genus Aneurinibacillus (80 % degree of confidence) and Genus Pantoea (47%) respectively. Isolate Ug Kiga_Mud1 is most likely to be a strain of Aneurinibacillus thermoaurophillus as indicated by a degree of confidence at 100%. Characterization for polymerase enzyme activity revealed that the crude protein extracted from compost isolates showed negative results when used in PCR while the crude protein extract from the geothermal hot springs showed positive results. The phenotypic characterization of Ug-Wampewo indicated that this putative novel bacterium is a gram negative coccobacillus with very large cream colonies on nutrient agar. The study recommends comprehensive physiological, biochemical and chemotaxonomic characterization of the novel putative organisms. In addition, a detailed assessment of the organisms is recommended in order to explore the potential applications of the novel putative bacteria in industry and biotechnology.
Assessment of event mobile application for livestock disease reporting in Karamoja

(Makerere University, 2018-11) Okori, Ongom Edward

The context of this study draws immensely from the global human and animal disease reporting perspectives, guided by the tripartite frameworks of WHO, FAO and OIE through their tools of IHR, EMPRES and PVS respectively down to the Pan African AU-IBAR, the East African Regional platform mandated by two Regional Economic Communities (RECs): IGAD and EAC. It further hinges on the premise that the success of disease reporting from the field depends on the competences to recognize clinical manifestations of the disease being reported, in addition to the use of diagnostic laboratories as key support to the diagnostic process. The high rejection of disease reports collected using EMA-i tool by field data collectors from Karamoja region of Uganda was the main objective of this study. The study purpose was to assess the impact of level of education and profession on the knowledge of EMA-i protocols. Forty one (41) data collectors from Karamoja, comprising of CAHWs, veterinary paraprofessionals and veterinarians were purposively selected and administered structured questionnaires for quantitative data analysis. In addition, a total of 18 key informants’ interviews including all the 7 Karamoja DVOs, 3 Karamoja based Veterinarians; 3 Officials from NADDEC/MAAIF, 3 paraprofessionals and 2 CAHWs leaders from Karamoja. In addition, 2 FGDs were held in Kotido district. The results from analysis of quantitative data, targeting education level, found out that out of the 41 reporters, only 1 primary reporter with education was knowledgeable while 5 were unknowledgeable; for Secondary education, 6 were knowledgeable and 4 unknowledgeable, whereas Diploma holders 6 were knowledgeable, and 1 not knowledgeable; while all the 18 University degree graduates were knowledgeable in most areas of the EMA-i protocol. Whereas for profession, 10 CAHWs were not knowledgeable and 9 knowledgeable while the 22 paraprofessionals and veterinarians were found to be knowledgeable in providing overall information on disease reporting using EMA-i. The information from the quantitative and qualitative data was triangulated with the results of EMA-i data from Karamoja during July 2016 to September 2017 that showed 45% of disease reports were invalidated. Basing on saturated opinion by KIs, it was established that most data by CAHWs are invalidated due to wrong diagnosis, indicative of their inadequacy in knowledge of the EMA-i tool, thus leading to the concluding that the level of education, is the main factor to quality of disease data.
Association between malaria preventive treatment regimens in pregnancy and plasmodium falciparum drug resistance mediating polymorphisms in Busia district, Eastern Uganda

(Makerere University, 2020-06) Nayebare, Patience

Intermittent preventive treatment in pregnancy (IPTp) with monthly sulfadoxine-pyrimethamine (SP) is recommended for malaria-endemic parts of Africa, but efficacy is compromised by resistance and, in recent trials, dihydroartemesinin-piperaquine (DP) has shown better antimalarial protective efficacy. We utilized blood samples from a recent trial to evaluate selection by IPTp with dihydroartemesinin-piperaquine or sulfadoxine-pyrimethamine of Plasmodium falciparum genetic polymorphisms that alter sensitivity to these drugs. The prevalence of known genetic polymorphisms associated with altered drug sensitivity was determined in parasitemic samples, including 375 collected before IPTp drugs were administered, 125 randomly selected from those receiving sulfadoxine-pyrimethamine, and 80 from those receiving dihydroartemesinin-piperaquine. For women receiving dihydroartemesinin-piperaquine, the prevalence of mixed/mutant sequences was greater in samples collected during IPTp compared to that in samples collected prior to the intervention for PfMDR1 N86Y (19.7% vs. 4.1%, p<0.001),PfMDR1 Y184F (73.7% vs. 53.6%, p=0.002), and PfCRT K76T (45.6% vs. 24.4%, p=0.002).Considering sulfadoxine-pyrimethamine, prior to IPTp the prevalence of all five common antifolate mutations was over 92%, and this prevalence increased following exposure to sulfadoxine-pyrimethamine, although none of these changes were statistically significant. The prevalence of two additional mutations associated with high-level sulfadoxine-pyrimethamine resistance was greater, but not significantly so, in samples collected during IPTp compared to those collected before the intervention (PfDHFR 164L: 9.3% vs.3.9%, p=0.07; PfDHPS 581G: 3.4% vs. 2.9%, p=0.73). Use of IPTp in Uganda selected for parasites with mutations associated with decreased sensitivity to IPTp regimens. Principal component analysis suggested that gravidity, parasite density, gestational age, participant age and treatment regimen contributed to the differences in prevalence of polymorphisms before and after IPTp.
Characterisation of integrons which potentially mediate dissemination of antibiotic resistance in enterobacteriaceae isolated from healthy community members

(Makerere University, 2020-10) Ato, Adera Doreen

Antibiotic resistance has become a global public health concern, by limiting the number of drugs used to treat bacterial infections. Antimicrobial resistance and multidrug resistance (MDR) therefore increase morbidity and mortality of people having bacterial infections in the community. Integrons are known as the genetic agents responsible for the capture and spread of antibiotic resistance determinants among diverse Gram-negative bacteria isolates. The activities of integrons have assisted the introduction of antibiotic resistance into genes pathogens and commensals of humans. Several studies have shown that integrons play a significant role in the spread of antibiotic resistance. This study examined the role of integrons in the spread of antibiotic resistance in Enterobacteriaceae from faeces of healthy community members. The antibiotic resistance patterns of 32 Escherichia coli and 8 Klebsiella pneumoniae isolates were determined by Kirby disk diffusion and conjugation was used to determine the transfer rates of antibiotic resistance. The 40 bacteria isolates were then tested for presence of integrons by polymerase chain reaction and the integrons present characterised by amplifying the integron variable region. The PCR amplicons of the integron variable region were then sequenced to determine the antibiotic resistance gene cassettes present. The isolates were found to have high resistance to beta-lactams (100%), sulphonamides (100%) and fluoroquinolones (88%). A high frequency of transfer ranging from 9×10-2 to 1.1×103 transconjugants per recipient E. coli J53 strain was recorded. The prevalence of class 1, class 2 and class 3 integrons were 100%, 35% and 0%. The integron cassette profiles were aadA1 (70%), dfrA17 (50%) and dfrA7 (10%) and they confer resistance to aminoglycosides and trimethoprim. The study revealed that 23 (58%) Escherichia coli and 2 (5%) Klebsiella pneumoniae isolates were MDR. The study also revealed that integrons with antibiotic resistance genes aadA1, dfrA7 and dfrA17are present in the isolates from healthy community members, thus they have a potential to capture genes which confer resistance to various antibiotics. The study recommends proper use of antibiotics, improved personal hygiene and sanitation to minimize the oral faecal spread of antibiotic resistance in the community. The study also recommends characterisation of transconjugants to determine which resistance gene cassettes are most disseminated.
Detection of carbapenem resistance due to outer membrane porin alteration in non carbapenemase producing E. coli and K. pneumoniae isolates from Mulago National Referral Hospital, Uganda

(Makerere University, 2022) Ndyagumizamu, Moses

Antimicrobial resistance is a global public health concern which compromises the use of antibiotics in the fight against microbes. Carbapenems are the most effective antibiotics for the treatment of clinical infections caused by Enterobacteriaceae, especially multi-drug-resistant strains. However, the emergence of carbapenem-resistant Enterobacteriaceae has become a major public health problem worldwide leading to limited treatment alternatives resulting in increased morbidities, mortalities and healthcare costs. This prompts for a need to clarify the resistance mechanisms as well as rapid and accurate detection of resistance if effective control is to be achieved. This study aimed at determining molecular mechanisms of carbapenem resistance in Escherichia coli and Klebsiella pneumoniae that are carbapenemase negative. A total of 87 isolates (47 E. coli and 40 K. pneumoniae) were screened for antimicrobial susceptibility by disc diffusion method. The 61 meropenem non-susceptible isolates were then screened for the production of carbapenemases, Extended Spectrum Beta Lactamases and AmpC enzymes phenotypically. The 28 carbapenemase negative isolates were then screened for outer membrane porin genes, followed by sequencing to detect mutations in the outer membrane porins. Out of 87 isolates, 28 (32.2%) were resistant to meropenem and 33 (37.9%) were intermediate. Among the 61 meropenem non-susceptible isolates, 36 (59%) were Extended Spectrum Beta Lactamase producers, 8 (13.1%) were AmpC producers, 23 (41.4%) were producing carbapenemases and 28 (45.9%) were carbapenemase negative. All the 28 carbapenemase negative isolates had at least one mutation in their outer membrane porin gene sequence that either resulted into a frame shift, deletion, insertion or substitution that led to early termination and production of truncated and non-functional protein, and/or produced an extended spectrum beta lactamase or AmpC enzyme. There is a high prevalence of carbapenem resistance in Mulago National Referral Hospital. Alteration of outer membrane porin proteins associated with production of extended spectrum beta lactamases and/or AmpC enzymes could have conferred carbapenem resistance in some of E. coli and K. pneumoniae isolates. There is need for the government to sensitize the public on proper antibiotic use. Healthcare workers should also prescribe appropriate antibiotics for effective treatment of carbapenem resistant infections.
Diet selection strategies of Grauer's Gorillas (Gorilla beringei graueri) in relation to nutritional benefits and exposure to hepatotoxic phytochemical in Mount Tshiabirimu Forest, Virunga National Park, DRC

( 2018-10-01) Kambale, Syaluha Eddy

The study aimed at understanding nutritional benefits and challenges encountered by the current population of Grauer’s gorillas on Mount Tshiabirimu within Virunga National Park. The relative concentrations of selected phytochemicals, nutrients and minerals were determined for eleven priority food plant species using multiple conventional methods. Results showed that all food plants differed with respect to concentrations of saponins, tannins and flavonoids (P<0.05). In addition, the food plants had higher levels of tannins followed by flavonoids, saponins and alkaloids respectively. Further, results demonstrated that food plants contained higher concentrations of carbohydrates followed by ash, dietary fiber, crude protein and lipids respectively. However, the carbohydrates concentration did not differ across food plants (P=0.68). In contrast, the concentrations of lipids, dietary fiber, ash and crude protein were different across food plants (P<0.05). Results also showed that, with the exception of Urera hypselodendron, all the most eaten food plants had the low protein-fiber ratio (below 1) whereas the less frequently eaten plants had high protein-fiber ratio (above 1) excluding Galium chloroionanthum. With respect to minerals, the concentrations of calcium, phosphorous, magnesium, sodium, potassium, copper and iron were different across food plants (P<0.05). Moreover, the concentration of potassium was higher than that of calcium followed by magnesium, phosphorus and sodium respectively across food plants. In addition, results demonstrated that the most commonly fed on food plants tended to have lower concentrations of alkaloids, flavonoids, magnesium, ash and lipids than the less commonly fed on plant species. Notably, the study revealed presence of hepatotoxic alkaloids, deficiency of lipids, sodium and copper in all food plants. The implications of the study findings on gorilla health are discussed.
Evaluation of nanoparticles and phages as candidate therapeutic agents for carbapenem resistant Klebsiella pneumoniae and Escherichia coli

(Makerere University, 2022-09) Ssekatawa, Kenneth

Centred on the current rate of antibiotics production and approvals, it is anticipated that by 2050 up to 10 million people could die annually due to multidrug-resistant (MDR) pathogens. One of the notable examples of antibiotic resistance is the evolution of carbapenem-resistant Enterobacteriaceae. The development of carbapenem-resistance (CR) is of great concern to the healthcare systems as there is no apparent next line of antibiotics to use against carbapenemase-producing (CP) Enterobacteriaceae. Among the Enterobacteriaceae, a high prevalence of carbapenem-resistance has been reported in Escherichia coli and Klebsiella pneumoniae. Indeed, the WHO expert committee on antimicrobial resistance included carbapenem-resistant Klebsiella pneumoniae and Escherichia coli associated with the severe urinary tract, bloodstream and lower respiratory tract infections as critical priority species. The ability to cause diseases is potentiated by the presence of virulence factors. The virulence factors influence the capacity of E. coli and K. pnuemoniae to infect and colonize different body systems. The coexistence of virulence and beta-lactamase encoding genes complicates treatment outcomes. To this effect, this study investigated the carbapenem resistance profiles of pathogenic E. coli and K. pnuemoniae in Uganda. Furthermore, nanomaterials and bacteriophages as alternative approaches to treat bacterial infections were evaluated. A retrospective cross-sectional study was conducted using 421 E. coli and 227 K. pnuemoniae archived clinical isolates collected from four Uganda tertiary hospitals between January and December 2019. The isolates were subjected to antibiotics sensitivity assays to determine phenotypic resistance. Five sets of multiplex PCR were performed to detect Carbapenem-resistance genes; DEC pathotypes virulence genes; ExPEC PAI; E. coli phylogenetic groups; and K. pnuemoniae capsular pathotypes. Silver and copper oxide nanoparticles were green synthesized using plant extracts. Chitosan was isolated from the banana weevils, mushrooms and Nile perch scales by demineralization, deproteination and deacetylation. Chitosan nanoparticles were prepared using the ionic gelation method. The AgNPs, CuONPs, Chitosan, and its derivatives were characterized by UV Vis spectroscopy, XRD, FTIR, DLS, SEM, EDX and TEM. Assessment of the antimicrobial activity of AgNPs, CuONPs and chitosan was done by the agar well diffusion method. Furthermore, E. coli phages were isolated from sewage by the enrichment method. The phage host range was determined by the spotting assay. Synergism of phages and nanomaterials in the elimination of biofilms was performed on a 24 well plate. Morphological and molecular characterization of phages were conducted. E. coli and K. pnuemoniae registered statistically similar phenotypic resistance (p-value = 0.9999). Molecular screening detected carbapenem-resistance determinants in 36.6% of the MDR isolates. K. pnuemoniae isolates recorded a genotypic prevalence of 43.2%, whereas E. coli scored 33.0%. Therefore, the prevalence of the carbapenem resistance genes was significantly higher in K. pnuemoniae (p-value < 0.0001). A significant variation between phenotypic and genotypic resistance was observed; however, the presence of resistance genes highly correlated with phenotypic resistance. A high-level carbapenem-resistance carriage was observed among pathogenic E. coli and K. pnuemoniae isolates. Among the 249 pathogenic E. coli, phenotypic resistance was found in 31.7% (79) isolates. For K. pnuemoniae capsular types, 34.9% (37/106) exhibited phenotypic resistance. Accordingly, compared to E. coli pathotypes, virulence factors and carbapenem resistance co-occurrence was significantly higher in K. pnuemoniae capsular types (p-value < 0.0049). Thus, the present study reinforces that carbapenem resistance is still a significant threat in Uganda as the phenotypic and genotypic resistance observed are statistically similar to those reported by earlier studies. Two novel phages (named UGKSEcP1 and UGKSEcP2) without any undesirable genes belonging to the Myoviridae phage family, Tequatrovirus genus, exhibited lytic activity against all the detected carbapenem-resistant E. coli pathotypes and four uncharacterized carbapenem-resistant K. pnuemoniae clinical isolates with extensive clear plaques. Furthermore, chitosan isolated from locally available materials exhibited antibacterial activity at extremely high concentrations (>3000 µg/ml), whereas the biosynthesized silver and copper nanoparticles had potent antibacterial activity at low MICs. Silver nanoparticles and silver-chitosan nanohybrid significantly reduced the phage viability but did not completely inactivate. A combination of phages and nanomaterials significantly reduced the planktonic and biofilm bacterial population density than the individual components. Furthermore, we found that the cocktail of phages and nanomaterials was more effective in preventing biofilm formation than treating. As the combinatory effect of phages and the nanomaterials was significantly higher than the individual components against carbapenem-resistant E. coli planktons and biofilms, therapeutic formulations containing phages and nanoparticles should be considered as alternatives to combat antimicrobial resistance.
Factors leading to misuse of developed water resources in the cattle corridor, Uganda

( 2018-10) Wanyama, Peter

Water is a fundamental resource in the cattle corridors worldwide. In Uganda, a number of water facilities have been developed in the cattle corridor. Despite this, there is persistent water shortage limiting cattle production. This could be due to misuse regardless of the WUCs. The factors that lead to misuse are however not well known. A cross-sectional study was conducted to: assess factors that influence water development; determine performance of WUCs and identify constraints faced by the WUCs. Primary data was obtained from twelve Focus Group Discussions, twenty four Key Informant Interviews using interview schedules and 317 randomly selected livestock farmers. Results indicated existence of WUCs in all study districts although their composition, roles and performance varied. The major roles of WUCs were: protection of water facilities (44.8%), repair of water facilities (25.6%), collection of user fees (16.4%) and training of users (10.1%). The WUCs were more effective in Nakasongola with positive association between sensitization and performance of WUCs (χ2 = 10.875; p = 0.000). Results also revealed WUCs best practices as: making and enforcement of bye-laws (65.3%), tree planting around water facilities (14.8%) regulating watering time (9.1%), construction of watering troughs (5.7%) and de-silting the water facilities (5.0%). Use of exchange visits (55.8%), drama and folk songs (18.9%), public media (13.2%) and extension workers (12.0%) were consequently identified as means of transferring WUCs’ best practices. Results further indicated: valley tanks (39.4%), valley dams (37.2%), roof catchment (14.8%) and ponds (5.4%) as the common water harvesting technologies used in the cattle corridor and distance of water facility significantly (χ2 = 66.259; p = 0.000) influenced watering frequency. It was further established that livestock water development was influenced by: number of livestock (27.8%), distance to the nearest water point (24.0%), production goal (15.5%), presence of alternative water source (14.8%), livestock watering frequency (11.7%) and water harvesting technology (6.3%). The results however indicated the factors that influence livestock water development varied within and between the study districts (χ2 = 40.232; p= 0.000). The constraints faced by WUCs were identified as: lack of fences on the water facilities (43.5%), poor cooperation of users (27.8%), lack of facilitation (20.2%) and limited capacity building (8.5%).The study recommends fencing and provision of security at water facilities, supporting exchange visits, scaling up sensitization of WUCs and giving incentives to motivate WUCs enhance their performance and ensure sustainability of water facilities in the cattle corridor.
Genotypes of Hepatitis B Virus in Sera of patients positive for HbsAg at Jinja Regional Referral Hospital between January-April, 2019

(Makerere University, 2022) Ssekibule, Robert

Hepatitis B virus infection is a serious disease that manifests clinically either as an acute hepatitis or in various chronic conditions causing approximately 650,000 deaths worldwide every year. About 10% of the Uganda’s population is chronically infected and are at risk of developing severe HBV liver related diseases. The HBV exists in different genotypes and each genotype has been shown to influence its clinical out and treatment differently. There is limited data about the circulating genotypes in different regions of Uganda where HBV is highly prevalent. In this study, the circulating genotypes of HBV in patients that reported to Jinja regional referral hospital between January to April 2019 were determined. In total, 102 stored HBsAg positive serum samples obtained from patients who reported to the hospital for HBV screening and testing between January to April, 2019 were included in the study. The DNA from the serum samples was extracted and tested them with PCR using primers targeting highly conserved pre-core/core region of all HBV genotypes. Of the 102 samples, 54 were successfully amplified as HBV which were then amplified by genotype-specific primers in order to determine the genotypes in the samples. Thirty five samples were successfully amplified by the different primer sets and the HBV genotypes identified as follows: D (n=12; 37.5%); A (n=8; 25.0%); E (n=2; 5.71%); C (n=1; 2.86%); B (n=1; 2.86%) and mixed genotype infections (n=11;31.43%). Representative amplicons of the different genotypes were sequenced and phylogenetic analysis carried out. All sequences of the amplicons were in agreement with the results of genotype-specific primers with the exception of genotype C which instead clustered with genotype A. In conclusion, genotype A and D were the most dominant genotypes in patients reporting to Jinja regional referral hospital. Proper management of HBsAg positive patients is recommended since Genotype D, the most dominant genotype in the region is associated with severe clinic outcomes.
Identification of suitable genes for RNAi silencing of aflatoxigenic apergillus flavus isolated from groundnuts in Uganda

( 2018-10-11) Ssekandi, Joseph Jr

Aflatoxins are lethal and carcinogenic secondary metabolites produced mainly by fungi of the Aspergillus genus, key among which is Aspergillus flavus. Previous research has shown that Ugandans are chronically exposed to these carcinogens and yet breeding A. flavus resistant varieties can be a sustainable solution to this problem. This study aimed at providing novel knowledge to groundnut breeders on which genes are suitable to silence in A. flavus so as to inhibit its growth using the RNAi technique triggered by dsRNA. Ten A. flavus isolates were isolated on Modified Rose Bengal Agar from 43 groundnut seed samples collected from Tororo, Soroti and Kamuli districts in Eastern Uganda and an Agraquant total aflatoxin ELISA kit was used to determine the amount of aflatoxins they produce. Only one isolate was non-toxigenic while others produced total aflatoxin ranging from five to over 2000 parts per billion. Thus there is diversity in A. flavus based on aflatoxin production ability. To determine the suitable genes for RNAi silencing in A. flavus, dsRNAs specific to 13 essential genes were transcribed in vitro using a T7 transcription kit. A. flavus spores were then exposed to these dsRNAs or sterile water for the controls and plated on ¼ strength PDA. Percentage spore growth inhibition was calculated and this spore growth inhibition data was subjected to ANOVA. Spore growth inhibition was significantly higher in all spores treated with dsRNAs targeting A. flavus essential genes in comparison to the controls treated with sterile water (df = 13, p < 0.001). Amongst the dsRNA treatments, silencing UBI, COH, 26s, ATP, PPK, IMP and ABC genes resulted in significantly higher spore growth inhibition than silencing GTT, HEL, PTRs, EXP, DRF and MOT genes (df = 13, p < 0.001). This study thus recommends UBI, COH, 26s, ATP, PPK, IMP and ABC as the suitable genes for RNAi silencing of A. flavus in vivo and also recommends a follow up study to establish the molecular basis for A. flavus diversity based on aflatoxin production ability.
Isolation of Staphylococcus Aureus and factors for colonization at a selected animal clinic in Kampala, Uganda

(Makerere University, 2021) Nabatta, Esther

Background: Staphylococcus aureus (S. aureus) colonizes numerous body sites and is one of the most frequently isolated bacteria associated with nosocomial infections worldwide. Treatment with antibiotics remains the cornerstone for control of these infections but this has been hampered by the emergence of resistant S. aureus which are shared pathogens of both medical and veterinary importance. Whereas comprehensive data on dynamics of body site colonization and antibiotic-resistant strains in animals and humans is available elsewhere, related information is very limited in sub-Saharan Africa including Uganda. Methods: A total of 303 swabs (156 swabs from out-patient dogs, 122 swabs from humans and 25 swabs from environmental surfaces) were collected and cultured for S. aureus. The isolates were subjected to a panel of 9 antibiotics and the drug resistance patterns determined. The isolates were then tested for the presence of the mec A and tet M genes using a conventional PCR. Results: The overall prevalence of S. aureus was 11.9% (36/303). Prevalence as per sample type was reported as 10.9% (17/156), 11.5% (14/122) and 20.0% (5/25) in pets, humans and the environment respectively. There was no significant difference in the prevalence of S. aureus isolated from pets, humans and the clinic environment. Nevertheless, monthly pet house cleaning frequency was a significantly associated with S. aureus prevalence in dogs (p=0.001). The highest resistance was observed against penicillin (100%), followed by resistance against tetracycline (70.6% to 100%). There was no resistance observed against gentamicin. The overall burden of MDR S. aureus was 75.0% (27/36). The tet M gene was detected in 47.2% (17/36) S. aureus isolates. Conclusion: Dogs had the highest prevalence of S. aureus compared to humans and the environment. There was no resistance observed against gentamycin making it the best drug of choice of drug to deal with S. aureus isolates in the event that there was an infection. Multidrug-resistant S. aureus colonizes both animate and inanimate surfaces and therefore these can be means of sharing MDR S. aureus. Resistance gene tet M was detected from S. aureus isolated irrespective of their phenotypic resistance and therefore care should be taken on use of antibiotics to avoid development of induced resistance. Lastly monthly pet house cleaning frequency was the only significant factor for colonization with S. aureus, suggesting that infections could be escalated by poor cleaning practices.
Knowledge and practice of dairy cattle zero grazing farmers in Kabale Municipality, South-western Uganda

(Makerere University, 2022) Kabagambe, Bernard

The study sought to examine the knowledge and practices of dairy cattle zero grazing farmers in Kabale municipality, Kabale district a south western region in Uganda. There has not been sufficient studies on farmers’ knowledge of zero grazing and its practices and the factors influencing it. The aim was to gain a better understanding of factors influencing zero grazing adoption, identify areas where zero grazing farmers lack in knowledge and assess how to boost their practices. Ultimately this study sought to subsequently make available information that could be used by extension workers, researchers, scientists and policy makers to further the promotion of zero grazing within the study area and beyond the confines of the study area. Data was collected using cross-sectional study design both qualitative and quantitative approaches were used in which the design allowed data to be collected at a single point in time to capture important aspects of the study. The researcher collected only primary data directly from the respondents of the selected farmers using a self-administered questionnaire and an interview guide to accomplish the study. The targeted population was 700 zero grazing cattle keepers within Kabale municipality from which a sample size of 255 was obtained. The main findings were that most of the dairy farmers in the study area demonstrated having basic knowledge about zero-grazing (80% of the respondents) but with limited awareness of vital technical skills in improved feeds and feeding; disease management; breed selection; and housing. The attitude of dairy farmers towards zero-grazing was mixed with positive perception attributed to profitability, space requirement, manure accumulation and disease control while negative perception was based on laborious nature of the enterprise, the time demanded and the cost incurred in starting up the use of the technology. It is therefore imperative that players in the country’s agricultural extension system need to boost efforts towards developing key transferrable knowledge packages and deliver such to dairy farmers on a regular basis to ensure the most efficient practices of dairy cattle zero grazing is a success in Kabale Municipality.
Molecular characterization, antibiotic susceptibility and phage sensitivity of Avian Pathogenic Escherichia coli isolated from colibacillosis cases

(Makerere University, 2021-02-22) Kazibwe, George

Avian Pathogenic Escherichia coli (APEC) cause extra-intestinal infections collectively known as colibacillosis, which result into significant economic losses for the poultry industry. The aim of this study was to characterize the prevailing Avian Pathogenic E. coli isolates and establish stocks of APEC lytic bacteriophages, for future development of cocktail products as alternatives to antibiotics in the management of avian colibacillosis. Fifty-six (56) glycerol stocks of Escherichia coli isolates obtained from chicken that had died of colibacillosis were analyzed for virulence, serogroup, phylogroup, antimicrobial susceptibility, and sensitivity to lytic phages. Escherichia coli characterization was done through phenotypic and multiplex PCR methods whereas antimicrobial susceptibility was done by Disk-diffusion method. Bacteriophage isolation and preliminary characterization was achieved using the spot assay and overlay plating techniques. All the 56 (100%) APEC isolates were resistant to at least one antibiotic while 50 (89.3%) isolates were resistant to at least three classes of antimicrobial drugs and were therefore designated as multi-drug resistant. All the selected five virulence genes were detected in the isolates with 39 (69.6%) of the isolates having at least one virulence gene. The virulence genes, ompT and iutA were the most frequent with 33 (58.9%) and 32 (57.1%) isolates respectively; while iroN least occurred in 23 (41.1%) isolates. Of the 56 E. coli isolates, 25 (44.6%) belonged to phylogroup A, 8 (14.3%) belonged to phylogroup B1, 1 (1.8%) belonged to phylogroup B2 and 22 (39.3%) belonged to phylogroup D. None of the 56 APEC isolates generated amplicons of sizes expected for the O1, O2 and O78 serogroups. Out of the seven isolated phages; two had the broadest host range with a combined lytic activity against 14 (25%) of the APEC isolates, coupled with stability to heat up to 60℃ and viability in pH ranging from 2 to 12. These results demonstrate that determining the presence of virulence genes can aid in confirmation of avian colibacillosis. The high level of resistance to the most commonly used antibiotics calls for a search for alternative treatment options like phage therapy in addition to antimicrobial susceptibility testing before treatment so as to limit economic losses due to ineffective treatment.
Molecular cloning and recombinant protein expression of 9-lipoxygenase gene from solanum tuberosum in pichia pastoris yeast cells

(Makerere University, 2021-03-05) Olengo, Alex

Lipoxygenases (LOX) are ubiquitous non-heme Iron-containing dioxygenases that catalyze the addition of molecular oxygen to Polyunsaturated Fatty Acids (PUFAs) such as Linoleic acid to form Oxylipins that possess anti-microbial activity. Synthetic fungicides have effectively controlled plant pathogenic fungi but their continued use has been detrimental to natural biological systems, and sometimes resulted in the development of fungal resistance. They also have undesirable effects on non-target organisms and foster environmental and human health concerns thus new biodegradable alternatives have to be investigated. The aim of this study was to generate a recombinant 9-Lipoxygenase protein for chemo enzymatic synthesis of Oxylipin based biodegradable fungicides. Golden gate assembly, a molecular cloning method that allows assembly of many DNA fragments into a complete piece using Type II s restriction enzymes and T4 DNA ligase was used to clone the large size of 9-LOX gene insert (2629bp). Three 9-LOX fragments were amplified from PET-28a-9-LOX recombinant plasmid vector and sub cloned individually into 3 intermediary level 0 vector pAGM35763. The recombinant intermediary plasmid vectors containing the 9-LOX fragments were used in a restriction-ligation reaction to assemble the 9- LOX fragments in the right sequence order into the destination plasmid vector pPICZaB. Chemically competent DH10B Escherichia coli cells were transformed with the ligation product, transformants selected and recombinant plasmid extracted and quantified. To achieve protein expression, the extracted recombinant plasmid vector pPICZaB was linearized and transformed into electrocompetent Pichia pastoris X-33 cells and grown on YPDS agar. Five Zeocin resistant transformant colonies were grown in BMMY media at 30°C and 250rpm for 4 days. Protein expression was induced by the addition of absolute methanol (0.5%) and methanol added every after 24h for up to 4 days to maintain protein expression. Analysis of protein expression from cell lysates was achieved by running an SDS-PAGE and Western blotting which showed a putative protein band of 97kDa representing 9-LOX protein. Results confirmed successful expression of recombinant 9-LOX protein in Pichia pastoris strain X-33. It is recommended that optimization studies on the yeast kex2 convertase and the a- secretion factor can be done to reinforce the secretion of recombinant Solanum tuberosum 9-LOX protein since the protein in this study was recovered from cell lysates.
Morphological and molecular identification of forensically important insect species (Diptera) in Kampala, Uganda using Mitochondrial Cytochrome Oxidase I Gene

(Makerere University, 2021-02-26) Mutesi, Lilian Doris

Insects attracted to corpses may provide important indications of the period post death otherwise called post-mortem interval (PMI), criminal event reconstruction, solving criminal cases, corpse movement and cases of neglect. The use of insects (Diptera) for PMI estimation in Uganda is of limited use since blowfly immature stages are not morphologically distinct. However, this has been solved by the use of molecular techniques which help identify insect species regardless of stage of development. They are quick, accurate and cost-effective. This study was conducted to identify insect species of forensic importance during decomposition stages of pigs (Sus scrofa domesticus) carcasses in Kampala, Uganda. Forensically important adult insect specimens were collected from outdoor (n=150) and indoor (n=120) carcasses and identified using taxonomic keys. Molecular identification of insect larvae was also done using Polymerase Chain Reaction (PCR) amplification of the mitochondrial cytochrome oxidase I partial gene followed by Restriction Fragment Length Polymorphism (RFLP), sequencing and phylogenetic analysis. The five stages of decomposition of the pig carcasses were categorized as: fresh, bloated, active, advanced and dry. The rate of decomposition in outdoor was faster than that in indoor environment. Three different families of Diptera; Calliphoridae, Sarcophagidae, and Muscidae were identified morphologically from both indoor and outdoor environments. The most abundant adult insect species was Chrysomya chloropyga (34.6%) and Chrysomya putoria (28.3%) for the outdoor and indoor environments, respectively. Sarcophaga bercaea was the least abundant adult insect species for both environments. All the species were present in the bloated and decay stages. Only Chrysomya chloropyga was found in the post decay stage. Other species were not observed in the post decay stage. Sarcophaga bercaea was not present in the bloated indoor.No insects were in the dry stage. Among insect larvae collected, Chrysomya putoria (32%) and Chrysomya chloropyga (48.1%) were the most abundant insect larvae from the indoor and outdoor environments respectively. Chrysomya albiceps (20%) and Hemipyrellia fernandica (10.9%) were the least abundant from the indoor and outdoor environments, respectively.Phylogenetic analysis of mitochondrial COI gene of insect larvae clearly clustered together and separated into four genetic clades of Calliphoridae flies (Chrysomya putoria, Chrysomya chloropyga, Chrysomya albiceps and Hemipyrellia fernandica). A fragment of the mitochondrial COI partial gene in this study displayed that DNA-based method can be used as supplemental means to morphological method for identification of the Calliphoridae as insect species of forensic importance in the different environments in Uganda. This concept should be extended to human corpses so that if they are consistent with results from Sus scrofa domesticus carcasses then this technique should be explored for homicide investigations in Uganda.
Perceived challenges of water scarcity on livestock production and pastoral community resilience in Qardho District, Somalia

Warsame, Ainab Mohamed
Phytochemical composition of Capsicum frutescens and its effect on growth, carcass, organoleptic and feed cost parameters of indigenous and broiler chickens

( 2022) Sebulime, Peregrine

Chicken meat is a major source of animal protein and its production has increased globally by 25% in the last decade mostly in Asia and Africa. However, due to the ever-increasing problem of antibiotic resistance, routine non-therapeutic antimicrobial use in chicken production systems is no longer considered a reasonable and viable practice. Natural products are among the preferred replacers of antibiotics for non-therapeutic uses such as promoting growth. In addition, the chicken meat produced from systems utilizing natural products is considered safer and protective of consumer health. Herbs and spices are important sources of natural products that can be used to improve growth and carcass traits of chickens in Uganda. Capsicum frutescens is among the common spices easily accessible but its use as an enhancer of growth and carcass traits in intensive chicken production systems has not yet been investigated in Uganda. This study aimed at establishing phytochemical composition and identification of dietary inclusion levels of Capsicum frutescens powder with positive effects on growth, carcass and organoleptic parameters of indigenous chickens and on growth and carcass traits of broilers. The phytochemical composition and antioxidant activity of C. frutescens powder were determined using conventional methods. A completely randomized design was used for each of three experiments for broilers and for each of the two experiments for indigenous chickens. The three experiments on broilers were differentiated by the durations of feeding diets enriched with C. frutescens. The three durations were: 13 days, 19 days and 24 days. Each duration represented the number of days prior to 59 days of age which was the age of slaughter. In each broiler experiment, three treatments defined by three dietary inclusion levels (1.1%, 2.2%, 4.4%) of C. frutescens powder were each randomly assigned to a group of forty-five broilers housed in three pens with each pen consisting of fifteen broilers. In addition, each experiment had a control group with forty-five broilers housed in three pens with each pen consisting of fifteen broilers. The control group received only the basal finisher diet. For the indigenous chickens, one of the experiments utilised cotton seed cake as the lipid source whereas sunflower seed cake was the source of lipids in the other. In the experiment where cotton seed cake was utilised, three treatments defined by inclusion levels (1.1%, 2.2%, 4.4%) of C. frutescens powder in diets, were each randomly assigned to a group of forty-five chickens housed in three pens with each pen consisting of fifteen chickens. The control group of this experiment had forty-five chickens housed in three pens with each pen consisting of fifteen chickens. The control group received only the basal diet. The other experiment of the indigenous chickens, where sunflower seed cake was utilised as lipid source, had two replicates instead of three and the treatments as well as number of chickens per replicate were similar to that earlier described for indigenous chickens. In both experiments of indigenous chickens, the chickens were aged 199 days at the start of the experiment and the duration of the experiment was 37 days. Results indicated that C. frutescens powder has several phytochemicals including alkaloids, coumarins, saponins, anthracenosides and reducing compounds. In addition, the total phenolic content, alkaloids and saponins were 587.75 mg GAE/100g, 6.8% and 23.5%, respectively. Further, the antioxidant activity scores based on DPPH radical scavenging activity at concentrations 100, 200, 400, and 500 ug/mL were 17.08, 20.75, 29.82, and 33.31%, respectively. Among broilers, diets formulated with 2.2% inclusion level and provided for 19 days had positive effects represented by significant increase in final body weight (1257.50±26.40g, P=0.007), carcass weight (786.18±22.39g, P=0.006) and gizzard weight (33.37±1.23g, P=0.02) by 14%, 15% and 12%, respectively relative to the control. Similarly, among indigenous chickens, diets with sunflower seed cake formulated with 2.2% inclusion level had positive effects demonstrated by significant increase in heart weight (4.00±0.33g, P=0.001), liver weight (15.30±0.80, P=0.04), proventriculus weight (4.25±0.14g, P=0.004) and intestine weight (82.50±3.58g, P=0.002) by 38%, 15%, 25%, and 21% respectively. Further, the 1.1% inclusion level increased intestine weight (68.00±2.17g, P=0.0002) by 26% whereas 4.4% inclusion level increased heart weight (3.60±0.21g, P=0.04) by 24% among indigenous chickens fed diets with sunflower seed cake. In contrast, the 4.4% inclusion level significantly decreased the body weight (912.50±32.05g, P=0.01), carcass weight (592.30±23.43g, P=0.04) and intestine weight (65.83±1.94g, P=0.001) by 12%, 10%, and 17% respectively among indigenous chickens on diets with cotton seed cake. Further, the 1.1% inclusion level decreased the liver weight (13.27±0.66g, P=0.02) by 14% whereas the 2.2% inclusion level decreased the heart weight (2.97±0.18g, P=0.001) by 19% among indigenous chickens on diets with cotton seed cake. Furthermore, results showed that none of the inclusion levels of C. frutescens had an effect on texture, taste, juiciness and overall acceptability of meat from indigenous chickens. The results of this study highlight that C. frutescens has several beneficial phytochemical compounds and high antioxidant activity for improving broiler growth and carcass yield. Further, an inclusion level of 2.2% in finisher diets for 19 days is acceptable since it has capacity to improve final body weight and carcass weight of broilers by 14% and 15% respectively relative to the control. Similarly, an inclusion level of 2.2% in diets with sunflower seed cake is recommended for indigenous chicken effective 199 days of age due to potential to improve carcass yield.
Porcine cyticercosis in selected districts of Uganda: Prevalence, pathology and relationship with human epilepsy

(Makerere University, 2018-12) Nsadha, Zachary

Taenia solium is a cestode that is zoonotic with the adult tapeworms found in human being (taeniasis) and larval stage cysts found in pigs (Porcine cysticercosis) or in humans (human cysticercosis). It is therefore a great disease of economic and public health importance. There are many instances when porcine cysticercosis has been found at meat inspection in parts in Uganda. No detailed study had been carried out on T. solium infections in Uganda. The aims of this study were to determine; the prevalence of porcine cysticercosis, risk factors for the acquisition of the infection, T. solium diagnosis and control, pathology of T. solium infection in naturally infected pigs and the spatial distribution of human epilepsy with taeniasis and and porcine cysticercosis. A cross sectional study was carried out the Lake Kyoga basin, international border points and national pig abattoir at Wambizi. Laboratory serodiagnosis was also carried out to determine the antigen sero-prevalence. Lingual examination was carried out for lingual prevalence. The lingual prevalence of porcine cysitercosis ranged from 4% to 12.9% for the areas of study in the Lake Kyoga basin. The sero-prevalence was found to be 25.3% (95% confidence interval 21.0- 30.0%) with district sero-prevalence ranging from 20.3-33.9%. The risk factors were; contaminated water used in the households and animal use, improper handling of infected pigs and pork for human consumption, human open-air defecations, inadequate knowledge about T. solium infections in the communities, inadequate control measures by the Veterinary Authorities, free-range pig husbandry. There was a positive spatial relationship between porcine cysticercosis, taeniasis and human epilepsy. In pig carcasses, the infection could be found in the non-traditional sites hence the lesion is more wide spread than what is covered in ordinary meat inspections. Cytokine TNF-α storm was observed in terminally-ill cysticercotic pigs. In conclusion the results indicate that porcine cysticercosis was prevalent and endemic in the in study areas, therefore a great problem. The risk factors for the acquisition of T. solium infections, which are the ideal areas of intervention and control of T. solium infection, exist in the study areas. A positive spatial relationship of porcine cysticercosis and human epilepsy occurs. I therefore recommend that deliberate interventions to reduce the T. solium infections in the pig and human beings need to be carried out. A study to find prevalence of human cysticercosis should be carried out the role of T. solium infection in the human human epilepsy. A study about the genetic diversity of T. solium in Ugnda should be carried out.
Prevalence and risk factors associated with occurrence of Camel Brucellosis in Mudug Pastoral Region, Somalia

( 2018-10) Mohamoud, Najah Mohamed

Brucellosis a zoonotic disease is primarily a disease of cattle and goats/sheep. Camels however are also infected by B. abortus and B. melitensis, which are from ruminants. A cross-sectional study was conducted to determine the prevalence and risk factors associated with brucellosis antibodies in camels in the selected districts of Galkacyo, Galdogob and Jariban in Somalia. Blood samples were collected from camels and screened for presence of antibodies using Rose Bengal Test. Seropositive samples were confirmed using I. ELISA. Additional information was collected from camel owners to identify risk factors like demographic characteristics such as sex, age, education of farmers as well as information about risks factors for transmission and maintenance of brucellosis in camels. Of the 420 camels sampled majority were females 86.4% (n=363) and adults 96.9% (407). Of these tested for brucellosis, 3.1% (n=13) were positive by RBPT. Seroprevalence was not significantly affected by sex, age, management system and source of camels (P > 0.05). When the seropositive samples were confirmed with I-ELISA, only 10/13 samples were positive, implying that there were 3/13 false positive results. Education level of camel owner, knowledge about brucellosis, camel abortion history or whether camels were herded separately or communally in village herds all had no significant association with occurrence of brucellosis. However, animals with abortion history showed higher prevalence (2.6%) compared to the ones without history of abortion (0.5%). It was concluded that brucellosis is still prevalent in camels in Somalia and therefore, there is need for increased surveillance in order to effectively control of the disease.
Prevalence and risk factors associated with occurrence of camel brucellosis in Mudug Pastoral Region, Somalia

(Makerere University, 2018-10) Najah, Mohamed Mohamoud

Brucellosis a zoonotic disease is primarily a disease of cattle and goats/sheep. Camels however are also infected by B. abortus and B. melitensis, which are from ruminants. A cross-sectional study was conducted to determine the prevalence and risk factors associated with brucellosis antibodies in camels in the selected districts of Galkacyo, Galdogob and Jariban in Somalia. Blood samples were collected from camels and screened for presence of antibodies using Rose Bengal Test. Seropositive samples were confirmed using I. ELISA. Additional information was collected from camel owners to identify risk factors like demographic characteristics such as sex, age, education of farmers as well as information about risks factors for transmission and maintenance of brucellosis in camels. Of the 420 camels sampled majority were females 86.4% (n=363) and adults 96.9% (407). Of these tested for brucellosis, 3.1% (n=13) were positive by RBPT. Seroprevalence was not significantly affected by sex, age, management system and source of camels (P > 0.05). When the seropositive samples were confirmed with I-ELISA, only 10/13 samples were positive, implying that there were 3/13 false positive results. Education level of camel owner, knowledge about brucellosis, camel abortion history or whether camels were herded separately or communally in village herds all had no significant association with occurrence of brucellosis. However, animals with abortion history showed higher prevalence (2.6%) compared to the ones without history of abortion (0.5%). It was concluded that brucellosis is still prevalent in camels in Somalia and therefore, there is need for increased surveillance in order to effectively control of the disease.

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