School of Biosecurity, Biotechnolgy and Laboratory Sciences (SBLS) Collection

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    Evaluation of Genetic Diversity among Glossina Fuscipes subpopulations from sleeping sickness foci in Uganda using Microsa Tellite DNA markers
    (Makerere University, 2008-01) P'Odyek, Patrick Abila
    Vector population genetic structure is rarely analyzed yet it is important in the understanding of the epidemiology of vector borne diseases and their control. In this study, patterns of genetic variation in the tsetse fly Glossina fuscipes fuscipes, the main vector of Human African Trypanosomosis (HAT) and Animal African Trypanosomosis (AAT) were assessed at five microsatellite loci. A total of 286 individuals were sampled from seven putative subpopulations in Uganda. Six of the subpopulations (Lumino, Kwapa, Osukuru, Namungalwe, Kaliro, and Serere) are sleeping sickness foci where the disease is endemic and the numbers of cases sometimes escalate. In one subpopulation (Ogur), G. f. fuscipes exists but active transmission is rarely observed. The mean number of alleles per locus for each of the subpopulations was relatively moderate ranging from 2 to 7 with generally low gene diversity (HE ranging from 0.2455 to 0.4376). Analysis of population structure with 21 possible pair wise comparisons revealed 12 significantly divergent pairs of which, Kaliro and Lumino populations had the least divergence (FST =0.0443, P>0.05) while Kaliro and Serere represented the highest divergence (FST =0.2288, P<0.0000). Analysis of molecular variance (AMOVA) indicated significant differentiation at all hierarchical levels between populations (FST = 0.1474, P<0.001), among populations within each group (FSC = 0.0313, P<0.001) and between the two groups (FCT = 0.1199, P<0.001). These data show that G. f. fuscipes populations are highly structured, with clearly defined northern and southern cluster that are separated by Lake Kyoga. These results are interpreted in terms of their implications for an area-wide integrated tsetse and trypanosomosis control/elimination strategy.
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    Evaluation of accuracy of the CD4+ T-cell counts using PIMA CD4 as compared to BD Facscaliber flow cytometry
    (Makerere University, 2010-05) Kafufu, Fred Bosco
    Introduction: Pima CD4 (Inverness Medical group 2009), a new method for enumerating CD4+ T-cells is affordable, technically simple, and economical in that it can use either electricity or battery. It is fully automated and thus, useful in remote settings. However, limited information on its performance exists in current literature. Thus, this study aimed at assessing the accuracy of Pima CD4 to BD FASCaliber flow cytometer (Becton Dickinson, Franklin Lakes and NJ) and precision of CD4+ T-cell counting using freshly collected capillary and venous whole blood samples, and establishing the types and frequency of errors incurred when using Pima CD4. This study reported the performance of the relatively new Pima CD4 laboratory test as compared to the more established FASCaliber flow cytometer in determination of the CD4 counts in HIV-infected individuals. The study addressed an important effort in up-scaling rapid, efficient and reliable means of management of HIV in resource limited setting such as in rural Uganda. Materials and methods Compared results of absolute CD4 counts obtained on replicate samples from 206 HIV-infected individuals (adult men and women) by Pima CD4 with those generated by BD FASCaliber flow cytometry at the Infectious Diseases Institute in Kampala, Uganda. Results Using venous whole blood, the mean CD4 counts were higher for BD FACSCaliber (422±220 cells/µL) but not significantly different (P=0.1289) from that for Pima CD4 (391±201 cells/µL). Results from the two machines were highly positively correlated (r = 0.96). The mean CD4 counts for BD FASCaliber using venous whole blood (422±220 cells/µL) were significantly higher (P=0.0116 at p=0.05 level) than for Pima CD4 using capillary blood (371±185 cells//µL). Further, the mean CD4 counts enumerated by Pima CD4 for venous whole blood (391±201 cells//µL) were higher than that in capillary blood (371±185 cells//µL) but not significantly different (P=0.3142). Further, within run precision demonstrated that the inherent imprecision of the Pima CD4 instrument is within the manufacturer’s claims and clinically acceptable limits. Also, between run precision demonstrated that the overall inherent imprecision of Pima CD4 instrument and due to other external variants is within manufacturer’s claims and clinically acceptable limits. Using venous whole blood the operator committed 24.3% errors with channel filling and reagent quality control being the most prominent. Likewise, when using capillary blood, the operator committed 13.6% errors with image and reagent quality control being the most prominent. Conclusions/recommendations Despite the few short comings, Pima CD4 maybe currently one of the suitable instruments for health centers and remote areas with limited access to CD4 testing centers. However, there is still need for proper and thorough training of the operators. Lastly, this study was absolutely done in laboratory environment. Thus, there is a need to evaluate the Pima CD4 in field conditions particularly in remote areas.
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    Genetic diversity of Plasmodium Falciparum infections at varying altitudes in South-Western Uganda
    (Makerere University, 2010-09) Nanyunja, Sarah
    Plasmodium falciparum is a highly polymorphic parasite. This sometimes results in a high genetic diversity in the population and high complexity of infections among individuals. Although some studies indicate a relationship between genetic diversity and transmission, there is not much information about the relationship between diversity and complexity infections at varying altitudes; a surrogate for transmission intensity. A retrospective cross-sectional study was done in two districts, Kabale (1700-2200m) and Rukungiri (1400-1600m), in South-western Uganda in 2007 from 10 villages at different altitudes to establish the relationship between diversity and complexity of infections at varying altitudes. A total of 1075 blood samples from members of the selected households were analysed by Paracheck-Pf and PCR. The diversity of Plasmodium falciparum circulating alleles was examined on the basis of the gene encoding merozoite surface protein 2 (msp2) using primers specific for the two allelic families, FC27 and 3D7. The overall prevalence of Plasmodium falciparum infections by PCR was 8.5% and 18.5% by Paracheck-pf. Examining the districts; in Rukungiri the prevalence was 13.3% by PCR and 29.9% by Paracheck-pf whereas in Kabale the prevalence was 0.7% by PCR and 0.5% by Paracheck-Pf. To assess the variability of circulating alleles, DNA fragments were grouped into classes of 40 base pairs. A total of 14 classes representing parasite populations were identified; 8 classes (60%) for 3D7 and 6 classes (40%) for FC27 family. Infections involving more than one allele in an individual accounted for 30%. The maximum number of alleles in an individual was 4 and the mean complexity of infections was 1.35 ± 0.60. The mean number of alleles (COI) at the different altitudes were compared, and a regression analysis revealed no significant association between the two p = 0.931). This study established the prevalence of Plasmodium falciparum infections in Kabale (<1%) and Rukungiri (~9%) and also demonstrated that no relationship exists between geneticdiversity and complexity of Plasmodium falciparum with increasing altitude. However, more studies are required in areas of higher malaria endemicities and a wider range of altitudes to evaluate this relationship further.
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    Effect of helminth infections and antihelminthic treatment during pregnancy on antibody response to Tetanus immunisation in women and their babies
    (Makerere University, 2010-11) Namatovu, Alice
    Uganda is still among the 46 countries that have not yet achieved neonatal tetanus (NT) global elimination target despite its adoption of the WHO strategy to vaccinate against tetanus during pregnancy. Immunisation of pregnant women with tetanus toxoid boosts immunity against the tetanus but a number of factors may influence the efficacy of tetanus toxoid immunisation in boosting immunity against tetanus. We examined the possible influence of helminth infections and antihelminthic treatment during pregnancy on levels of antibodies against tetanus toxoid. The study was conducted using archived serum samples that were collected from infants at one year of age and their mothers who were enrolled in the Entebbe Mother and Baby Study (EMABS) during their pregnancy in a randomised, double-blind, placebo-controlled study of deworming during pregnancy with albendazole or praziquantel. Levels of total IgG and subclasses IgG1, 2, 3 and IgG4 against tetanus toxoid were measured by ELISA for women during pregnancy and at delivery and for their offsprings in cord blood and at the age of one year. The data was analysed using median, ranksum test, Chi-square test, signrank test and Spearman’s test. The prevalence of helminth infections among the enrolled pregnant women was 65% with most of them having light infection intensity. The predominant worms were hookworm (40%), Schistosoma mansoni (17%) and Mansonella perstans (20%). Sixty six percent (66%) and 90% of the women had detectable total IgG levels at enrolment and delivery respectively. The median total IgG, IgG 1, 2, 3, and 4 antibody levels at enrolment were 4.4 (0 - 15) IU/ml, 731 (266 - 1378) µg/ml, 64 (28 - 148 ) µg/ml, 188 (0 - 424) µg/ml and 57 (0 - 376) µg/ml, respectively. There was a significant increase in total IgG and all its isotypes at delivery except IgG3 (p =0.374). The cord blood antibody levels positively correlated with delivery antibody levels but not with levels at one year. Antibody levels for infants at one year were lower than the cord blood levels for all the antibodies. Maternal helminth infections and antihelminthic treatment had no significant effect on all the maternal and offspring’s antibody levels except for IgG2 where albendazole treatment during pregnancy in hookworm infected women was associated with reduced IgG2 levels in infants at one year. However, since it was not consistent with our study hypothesis and not related to any other findings, it may have been due to individual variation in immune system or due to other factors which were not part of this study such as nutrition. Hence helminth infections and antihelminthic treatment during pregnancy did not have effects on maternal and infant response to tetanus immunisation likely to be of significant public health importance. This conclusion is based on results obtained from a cohort of pregnant women where the majority had light helminth infection intensity. Further studies with a cohort of women with heavy helminth infection intensity are necessary to explore effects of helminth infections and antihelminthic treatment on immune response to tetanus immunisation. Studies to explore how infancy and childhood helminth infections and their treatment could affect the immune response to tetanus immunisation in clinical controlled trials are also needed.
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    Assessment of health laboratories in handling epidemics of public health importance in Kampala District
    (Makerere University, 2011) Kabasa, William M.
    Background: Scanty information is available on the extent to which health laboratories in Uganda are involved in managing disease epidemics. According to Central Public Health laboratories report, laboratories in health facilities, including health centers in urban areas with high population densities have a special role in reducing risks, containing emergencies and responding to outbreaks. Moreover health facilities that are affected or exposed to risks, emergencies or outbreaks and are unprepared may pose additional or more serious risks to the communities they serve. This study assessed the level of preparedness of health laboratories in handling epidemics of public health importance in Kampala District. Method: A standardized, semi-structured questionnaire was used to collect information from (30) private health facilities and eight (8) public health center threes (HC3s) making a total of thirty eight (38) laboratories. The collected data was coded and analysed using a Stastical Package for Social Scientists (SPSS). Results: Seven core capacities were analysed where on staffing, laboratory based staff such as microbiologists, laboratory technologists, laboratory technicians and microscopists were 10.0%, 10.4%, 11.1%, and 10.4% , while staffs who got training for any specific disease outbreak of concern were highest in public health labs at 60% and lowest in private labs at 45.2% and those who never trained were highest in private at 52.4% and lowest in public labs at 30%.; Parasitology and serology scored highest at 74.1 %, in the tests performed while mycology scored the lowest at 1.9 %. Biosafety level 2 (BSL2) was more commonly used at 50% while BSL 3 was least used at 9.3%, moreover 25.9 % of respondents implemented Biosafety measures. Handling of disease outbreaks was more in private laboratories at 27.5% and lowest in public labs at 20%. As regards to equipment servicing, 60 % was found to be in public labs while private labs had 58.1%. Results referrals, with reference labs, was lowest in private labs at 83.7% while highest in public labs at 90%. Conclusion: This study suggests that laboratory requirements for management of disease outbreaks were the same for both private and public health units. The status of health laboratories in general laboratory safety management and good laboratory practices were inadequate. EQA practices in the study showed that results for laboratory disease diagnosis in the surveyed health units were accurate and reliable.
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    Potential of cell cycle genes arath cycd2 and musa cycd2 for the improvement of transformation and regeneration efficiency of banana (cv “sukali ndiizi”)
    (Makerere University, 2011-03) Samukoya, Clara
    Available techniques for the genetic transformation with important genes could overcome some of the agronomic and environmental problems limiting conventional breeding of bananas. Although it is possible to transform bananas, broad application of the technology is limited because of the low overall efficiency and lack of reliability of the technique. This research reports on the potential of CycD2 genes to improve transformation and regeneration efficiency of banana (cv. “Sukali ndiizi”). Two genes Arath CycD2 and Musa CycD2 were evaluated for the cell cycle modification of the embryogenic cell suspension that is conventionally used in banana genetic engineering at the National Biotechnology Centre, Kawanda. The UidA (GUS) gene was used as reporter to establish transient transformation efficiency. The GUS reporter gene, which was used for quantification of transformants was therefore, fused with each of the CycD2 genes in the binary vector, pC1305.1. The Cauliflower mosaic virus 35S promoter was used to drive both CycD2 and the GUS reporter genes. Results indicated that cell cycle genes could significantly increase the competence of banana cells for uptake and integration of foreign genes. The Gus assay analyses of transformed cells showed a success rate of 80% to 90% for all the constructs including the control transformed with the empty vector without CycD2 gene. To assess whether the CycD2 genes could improve the regeneration efficiency of “Sukali ndiizi”, the transformed cells were cultured on selection media and the hygromycin resistant colonies developed into shoots. The gus assay of the regenerants showed that the genes were expressed in different parts of the plants (roots, corm and leaves). The PCR analysis of DNA from these shoots indicated that Musa CycD2 and Arath CycD2 genes significantly improve the regeneration of transgenic “Sukali ndiizi” cells. The regeneration effinciency of the embryogenic colonies of CycD2 genes (47%-62%) was much higher than that of the control without CycD2 (18%). The results show that “Sukali ndiizi” cells are highly competent and transformable by Agrobacterium mediated transformation system and CycD2 genes have the potential to significantly improve regeneration efficiency of “Sukali ndiizi” cells”. This study contributes to the current information about improvement of transformation and regeneration efficiency of bananas and highlights the potential of CycD2 genes in the improvement of regeneration of transgenic plants.
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    Detection and characterization of human influenza A virus isolates from patients attending Kayunga and Mulago Hospitals in Uganda
    (Makerere University, 2011-09) Kaira, Blanche Byarugaba
    Human influenza A viruses are known to cause severe illnesses and fatalities. Therefore, their continuous surveillance is crucial for the early detection and appropriate interventions through treatment therapies and vaccination. The aim of this study was to detect and characterize human influenza A virus isolates from Kayunga and Mulago hospitals in Uganda. A total of 450 human respiratory samples were collected. All samples were screened for influenza A virus using a One Step RT-PCR Qiagen kit and M gene specific primers from Applied Biosystems, UK. A volume of 100 µl from each of the samples that tested positive for Influenza A viruses were cultured on MDCK (Madin Darby Canine Kidney) cell line passage 2 in T25 culturing flasks. The isolates that showed cytopathic effect (CPE) were subjected to immune-fluorescence assay (IFA) using a Light Diagnostics ™ Influenza A and B DFA kit. The samples confirmed positive by IFA were then sub-typed by One Step RT-PCR Qiagen kit using H1N1 and H3N2 specific primers from Applied Biosysems. This was followed by whole genome DNA sequencing of PCR products using the Illumina Genome Analyser IIe. There were 51 positive samples for influenza A viruses detected by One Step RT-PCR. The viruses from the samples all showed CPE when cultured on MDCK cell line and were further confirmed positive for influenza A viruses by IFA. The prevalence of the influenza A viruses was 11.3%. The participants who were of the 6 months to 5 years age-group had the highest infection rate, 80%. The infection rates were highest during November 2008 and the Mulago site registered more positive cases. All viruses detected were of the H3N2 subtype. The influenza A viral genes, PB2, PA, and NP, had significant amino acid changes that have been reported to be associated with host transmission and low virulence. All study isolates were of different strains that had nucleotide diversities by each of the eight genes. The human influenza A H3N2 viruses detected were already existent worldwide. Their gene sequences were 99% similar to those of other strain sequences of viruses detected in 2008 and submitted in the genbank database. The study findings indicated the presence of influenza A H3N2 viruses with a prevalence rate of 11.3% in the Ugandan human population. All the study strains were already existent worldwide
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    Evaluation of Bacillus thuringiensis crystal (CRY6A) and carica papaya cystatin toxins against the banana weevil (Cosmopolites sordidus) using a novel diet and construction of a plant transformation vector with two stacked toxin genes
    (Makerere University, 2011-10) Bakaze, Elyeza
    The banana weevil (Cosmopolites sordidus) is a major pest of banana, plantain and Ensete. Despite the existence of control strategies that combine cultural, biological, use of botanical extracts and chemical control methods, banana weevil damage and the associated crop losses have persisted. Although resistance is available in wild banana, conventional breeding is not feasible. Genetic engineering as an alternative strategy to conventional breeding or in combination has been suggested for the provision of weevil resistant banana varieties to farmers, especially with maintained taste and cultural preferences. However, this has been hampered among other experimental factors, by the lack of an artificial diet to perform rapid bioassay experimentation to screen resistant banana cultivars and potential candidate proteins for a transgenic approach. This research modified an artificial diet in which banana weevil neonates were able to develop up to adult stage in 48 days compared to 36 days on natural banana stem diet. It also reports on the use of this diet to evaluate resistance in banana germplasm and the in-vitro efficacy of Bacillus thuringiensis Cry6A as well as Carica papaya cystatin (CpCYS) toxins against C. sordidus. The laboratory produced recombinant Cry6A and CpCYS proteins and their use in the diets is described. The modified artificial diet allowed preliminary evaluation of efficacy and potential of these two genes in the control of banana weevil. Fifty percent mortality (LD50) of neonate larvae was observed at 0.24 parts per million and 0.15parts per million for Cry6A and CpCYS respectively. Both genes were cloned together into a plant expression cassette to create a plant transformation binary vector. This research therefore modified an artificial diet that sustained 70% of banana weevil through all growth stages and using this diet established the efficacy of two recombinant proteins; a basis for developing transgenic bananas with improved resistance against banana weevils.
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    Gene transcript analysis in drought stressed ‘AAA’ and ‘ABB’ bananas using next generation sequencing technologies
    (Makerere University, 2012-08) Nyine, Moses
    The main purpose of this study was to determine differences in gene expression and allelic heterozygosity in two banana genotypes, Mbwazirume ‘AAA’ which is susceptible to drought stress and Cachaco ‘ABB’ which is tolerant when exposed to drought stress. This was achieved using data from 454 and Illumina sequencing platforms. Drought stress tolerance in bananas has been associated with the B genome but no linkage between gene expression and drought had been illustrated. This has been in part caused by lack of a reference genome/reference transcriptome making it hard to conduct gene expression studies. In this study a reference transcriptome was generated using 454-pyrosequencing technology that comprises of 21201 contigs of lengths ranging between 500-4000 bp. During the course of this project the DH Pahang genome from the CIRAD team was accessed, which together with the reference transcriptome enabled a comprehensive analysis of gene expression and allelic heterozygosity in the two genotypes. Mbwazirume and Cachaco share many genes but what differs is the expression level of these genes. Many genes that are associated with drought tolerance were up-regulated in Cachaco and down-regulated in Mbwazirume. In cases where both genotypes showed down-regulation such as expression of aquaporins, Cachaco maintained a higher level than Mbwazirume. Transcription factors such as MYB44, MYC4, NAC, bZIP, DREB1 and DREB3 were up-regulated in tissues of Cachaco and down-regulated in Mbwazirume. The same trend was observed for genes for antioxidant enzymes and cell cycle regulating proteins. The ability of Cachaco leaves to express high levels of SIC-SGP with reduced expression of LSRP and chlorophyll catabolic enzymes would help it retain more green leaves under dry conditions. Many SNPs were detected in Cachaco, with majority being heterozygous. This was attributed to the presence of two copies of the B genome. It is possible that some of these alleles affect gene activity and efficiency of gene transcription, which results in differences in response to drought stress.
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    Serotypes of foot and mouth disease virus in African buffaloes and cattle in Queen Elizabeth National Park
    (Makerere University, 2014) Ruhweza, Simon Peter
    A cross-section study was done in Queen Elizabeth National Park (QENP) to determine the FMDV serotypes circulating in buffaloes and cattle. Serum and oro-pharygeal fluids (probang samples) were collected from African buffaloes (n=36) and cattle (n=114) from August 2011 to June 2012. Serum was screened using Priocheck FMDV NS ELISA and oro-pharyngeal probang samples were screened using Real-Time PCR. Serotype specific antibodies were determined by solid phase blocking ELISA (SPBE) and Virus Neutralization Test (VNT). The VP1 gene of positive PCR probang samples was amplified and genetic relatedness determined by construction of a phylogenetic tree. On Priocheck NS ELISA; 20/114(18%) cattle and 26/36 (72%) buffalo sera were positive. On SPBE; antibodies against serotypes; O (5/7; 71.4%), SAT 2 (1/6; 16.7%) and SAT 3 (2/8; 25%) in cattle and serotype; O 1/22(4.5%), A 1/8 (12.5%), SAT 1 (4/20; 20%), SAT 2 (5/29; 17.2%) and SAT 3 (3/23; 13%) in buffalo were detected. By virus neutralization test only antibodies against serotype; O 3/23 (13%) in cattle were detected, while in buffaloes it was only antibodies against SAT 2 (10/29, 35.4%) and SAT 3 (2/23, 8.6%) were detected. Real-Time PCR detected positive 3/20(15%) in cattle and (16/26) 61.5% in African buffalo. The VP1 gene, responsible for coding the major antigenic determinant of FMD virus, was used to characterize the SAT 2 sequence (UGA 11/13) that was got from buffalo. The phylogenetic analysis showed that it belonged to the East African buffalo lineage and was closely related to the previously isolated SAT 2 FMD sequences Buffalo 6 QE with pair wise identity of 83%, and Buffalo 10 QE with pair wise identity of 82%. The findings confirmed that SAT serotypes are found among African buffaloes in QENP. This study also showed that the circulating FMDV serotype antibodies in buffaloes and cattle were not the same since only antibodies against serotype; O were found in cattle sera while serotype; SAT 1 and SAT 2 in buffaloes by VNT.
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    Seroprevalence and risk factors for brucellosis in cattle and humans in selected districts of Jimma zone, Southwest Ethiopia
    (Makerere University, 2014-09) Bashahun, Gebremichael Dirar
    A cross-sectional study was carried out in two selected areas of Jimma zone from February 2014 to May 2014 to determine sero-prevalence of Brucellosis and associated risk factors in cattle and humans. A total of 348 blood samples from cattle (174from Chora Botor district and 174 from Jimma town) and 48 human blood samples (24 from Chora Botor and 24 from Jimma town) were collected. The collected blood samples were screened using Rose Bengal Plate Test (RBPT)and positive ones were further subjected to Complement Fixation Test (CFT) for confirmation. Statistical analyses were performed using SPSS version 20 software. The overall sero-prevalence of Brucellosis and demographic characteristics of the herd owners was performed using descriptive statistics. Chi-square test was used to determine association between explanatory variables and outcome variables.Results in cattle showed overall sero-prevalence of 1.4% and 0.3% as tested by RBPT and CFT, respectively. In Chora Botor district and Jimma town, the sero-prevalence of Brucellosis in cattle was found to be 1.1% and 0.6%; and 1.7% and 0% as tested by RBPT and CFT, respectively. In humans the sero-prevalence was 2.1% and 0% by RBPT and CFT, respectively. Retained fetal membrane was significantly associated with sero-positivity of Brucellosis in cattle (p=0.019). No statistically significant variation in prevalence of Brucellosis was found among the different location, age, sex, and herd size, breed and management system of the animals. The majority (97.6%) of the respondents had no knowledge and awareness about the zoonotic importance of Brucellosis. This warrants awareness creation about the zoonotic importance of the disease and transmission modes particularly to those high risk groups to minimize contact with animals and their products. Occurrence of the disease in both humans and animals in the study area warrants effective control using the One Health approach which is the most constructive strategy embraced.
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    Occurrence of cassava mosaic geminiviruses and cassava brown streak viruses in cassava and wild plant species in Uganda
    (Makerere University, 2015) Adero, Joanne
    Cassava (Manihot esculenta crantz) is an important food and cash crop worldwide. Currently cassava brown streak disease (CBSD) caused by Cassava brown streak ipomoviruses (CBSVs) and cassava mosaic disease (CMD) caused by Cassava mosaic geminiviruses (CMGs) are the major constraint to cassava production in East Africa. Occurrence of these viruses in alternative hosts is unknown and therefore potential risk posed to cassava cultivation. A survey was conducted in five agro ecological zones of Uganda, in 24 cassava growing districts to determine plant species that may be alternative hosts for these viral pathogens. Leaf samples plant species with virus-like symptoms found within or adjacent to cassava fields were picked and analyzed. Overall CBSD mean incidence for the 24 districts was 38.4%. Kayunga district had the highest incidence (100%), while Soroti had the lowest incidence at 4.7% and two districts did not have CBSD. CMD mean incidence was 13.4%, with Busia having the highest incidence at 46% while Kamuli had the lowest incidence at 0.7% and five districts did not have CMD. Nucleic acids were extracted from both cassava and suspected alternative plants, and tested for CMGs by conventional PCR and CBSVs by two-step RT-PCR and analyzed by agarose gel electrophoresis. Twelve (12) wild plant species were positive for CMGs with nine (9) plants positive with ACMV and three (3) plants positive for EACMV-UG. Ten (10) plants were positive for UCBSV and one (1) was detected with CBSV. PCR products were purified and quantification and Amplicons were sequenced in both directions. All nine ACMV sequences, three EACMV sequences and one CBSV sequence obtained from non-cassava host plants had significant similarities with sequences in the gene bank with sequence having 89–100% nucleotide sequence identity with cassava isolates from East and West Africa. Plant species identification was done for confirmed alternative host plants for CMGs and CBSVs, and plants were identified into seven species namely Ageratum conzyzoides, Asystasia gangetica, Solanum incanum, Leonotis nepetifolia, Hewettia sublobata, Erythrina abyssinica, and M. glaziovii. These results provide definitive evidence for the natural occurrence of cassava viruses in plant species besides cassava, acting as reservoirs for the viruses. This therefore demands appropriate measures to safeguard cassava production in Uganda.
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    Evaluation of process procedures of selected medicinal plants used for treating malaria fevers in Tororo district
    (Makerere University, 2015-03) Odda, John
    Background: Despite widespread utilization of medicinal plants globally, there is insufficient documentation regarding their efficacy and standard processing procedures (SPP)-leading to products with variable levels of active substances and unpredictable pharmacological action. The purpose of this study was to determine if selected antimalarial herbs from Tororo, Uganda, are processed as per WHO requirements, factors influencing the manner of preparation and to determine efficacy, quality and levels of active principles of products when SPP are not followed. Methods: Cross-sectional studies were used to document knowledge, attitude and practices (KAP) of herbalists regarding SPP. Laboratory assays were used to determine antiplasmodial activities and safety of the study botanicals. Method (OPQ/STP/FP-3144-1/5) at Med Pharm Uganda was used for phytochemical analyses, herbal Plumbagin and Phyllanthin contents as well as chemical and physical stability parameters. Standard laboratory methods were used to determine efficacy, biological and toxicological parameters of herbal powders in a six month stability study. Results: From 106 herbalists, 100 had scanty knowledge about SPP while 100% opted not to label their herbal products. Furthermore, herbalist practices from plant identification to storage are still below WHO standards. Plumbago zylenica extracts processed by indigenous knowledge (IK) displayed high in vitro antiplasmodial activity on chloroquine sensitive (3D7) strains (IC50 of 4.2 - 4.3μg/ml). The in vitro antiplasmodial activity of IK prepared Phyllanthus amarus extracts was low compared to those prepared according to WHO (IC50= 12.15μg/ml and 3.533μg/ml respectively). On the other hand, Phyllanthus amarus shoot extracts exhibited significant in vivo antimalarial activities targeting trophozoite stage of the parasite. Plumbagin content in Plumbago zylenica IK processed root was significantly higher than those meeting WHO requirements (0.38 ± 0.015μg/ml and 0.35 ± 0.01/ μg/ml respectively), P<0.05 while P. amarus IK processed shoot contained less phyllanthin compared to those meeting WHO requirements (1.972 ± 0.24μg/ml and 4.286 ± 0.03/ μg/ml respectively), P<0.05). Both P. zylenica root and P. amarus shoot unpreserved powders are stable for 6 months at 25±5oC and RH of 65 ± 5%. However, the quality of the IK processed herbs is low due to unacceptable levels of aflatoxins. Conclusion: This study has documented KAP regarding herbal SPP in Tororo. The Ugandan Phyllanthus amarus shoot has demonstrated in vivo antimalarial activity against Plasmodium berghei trophozoites. Both P. zylenica root and P. amarus shoot unpreserved powders are stable for 6 months at 25±5oC and RH of 65 ± 5%. Factors influencing the manner of preparation included lack of a proactive herbalist Association, refresher courses and negative attitudes on (SPP). Recommendations: It is recommended that MoH in collaboration with the Ministries of Youth, Gender and Culture, Agriculture and Forestry as well as researchers to address issues raised in this study.
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    Production methods, physico-chemical properties and microbial quality of bongo, a fermented milk product produced in Pallisa District
    (Makerere University, 2015-03) Gulaita, Norah
    Production of fermented milk products using traditional methods has been carried out since time immemorial across the world. One of such products is bongo, a yogurt-like traditionally fermented dairy product produced and sold in Uganda. The quality of bongo sold in Uganda is not known and this has greatly affected its commercialization. This study was carried out in Pallisa district to characterize the production of bongo by documenting the processing methods, determining its physico-chemical, hygienic quality and the lactic acid bacteria diversity therein. A survey using focus group discussions to identify renowned traditional bongo processors (TBPs), key informant interviews with the TBPs and observations were carried out. Household bongo samples were collected from the bulk fermentation vessels of TBP and analyzed for pH, titratable acidity, total solids and fat content. Total plate, coliform, yeast and mold counts were enumerated on standard media. The lactic acid bacteria in bongo were isolated on agar and then characterized using standard phenotypic traits. The study revealed that two types of bongo are processed in Pallisa district coined ‘beverage’ bongo referring to sour milk that is consumed as a drink and ‘vegetable’ bongo referring to sour butter milk used in cooking traditional sauces. Beverage bongo was made from boiled (pasteurized) cow milk whereas the vegetable type was made from (fresh) cow milk. The average pH, titratable acidity, total solid and fat content for drinkable bongo were 3.86 ± 0.34, 1.60 ± 0.53, 7.14 ± 0.26% and 0.83 ± 0.17% whereas those for vegetable bongo were 3.71 ± 0.12, 1.75 ± 0.13, 7.84 ± 0.19% and 0.79 ± 0.35% respectively. The mean total coliform counts were 2.11 ± 2.34 log cfu/ml and 4.83 ± 5.54 log cfu/ml for beverage and vegetable bongo respectively. The yeast and mold counts were 4.29 ± 4.25 log cfu/ml and 4.49 ± 4.35 log cfu/ml for drinkable and vegetable bongo respectively. The lactic acid bacteria identified in bongo belonged to three genera namely Enterococci (81%), Streptococcus (15%) and Lactobacillus (4%). The study reported high microbial contamination of bongo indicating poor processing hygiene thus rendering the product unsafe for human consumption. Therefore, cross contamination of bongo during its production should be minimized.
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    Incidence, yield loss and economic injury levels of key insect pests of hot pepper (scotch bonnet) in major growing Districts of Uganda
    (Makerere University, 2015-10) Muzira, Fred
    Hot pepper, scotch bonnet is considered among the high valued nontraditional cash crops grown by farmers in Uganda. Its production however is constrained by insect pests, diseases coupled with poor agronomic practices. Limited information exists regarding the level of occurrence of insect pests and their effects on hot pepper yield in Uganda. This is important for developing sound, environmentally friendly and cost effective management practices. This study was therefore conducted to (1) determine the occurrence and distribution of insect pests of hot pepper in major growing districts of Uganda, (2) Establish the relationship between infestation/damage levels of insect pests and fruit yield of hot pepper, and (3) determine the action threshold and economic injury levels of the two most prevalent insect pests of hot pepper. Biological monitoring surveys were conducted to establish the occurrence of insect pests in 6 major hot pepper growing districts of Uganda. The districts are Mukono, Wakiso, Kiboga, Hoima, Luwero and Mpigi. A total of 82 farmers were visited during the study. Data was collected by throwing four 2x2m quadrats; 3 along a diagonal transect and the other at the furthest point from the diagonal. All the enclosed plants were inspected for possible life stages of the insects or their damage. Results of the surveys indicated that fruit flies (Ceratitis capitata), aphids (Aphis gossypii), whiteflies (Bemisia tabaci), bollworm (Helicoverpa armigera and Spodoptera litoralis) and thrips are the most abundant and prevalent insect pests of hot pepper. They occurred in all the visited districts. Fruit flies had the highest incidence (37.1%), followed by aphids (26.1%), whiteflies (23.7%), and bollworm (3%). Thrips had the lowest occurrence (1.7%). The effects of location and cropping system on insect pest occurrence were significant (P<0.05). The occurrence of insect pests (majorly sucking pests) was lower in mixed cropping system compared with monocropping. To compare infestation levels/damage and yield losses caused by the pests, a repeated on station trial was conducted. Five different spray schedules were used to create variable pest levels. The trial was laid out in a Randomized complete block design (RCBD) with three replications. The five spray schedules included; 1) application of insecticide once a week to 50% bud initiation; 2) Weekly sprays from bud initiation to 50% fruit initiation; 3) weekly sprays from 50% fruit xii initiation to first harvesting; 4) Weekly sprays throughout the growing period of the crop, and 5) untreated control. The population dynamics and damage of the insect pests were recorded over the growing period. Fruit yield was also recorded at first harvest. Results indicated that on average, insect pests significantly reduced yield by 63% if compared to treated plots. Fruit damaging insect pests caused the highest amount of yield losses (55%). Spraying starting at fruit initiation was as effective in reducing yield loss as spraying weekly throughout the growing season. In fact, the study revealed that chemical control was more profitable when initiated at fruit initiation to target the fruit damaging pests than when used throughout the growing period. To determine the economic injury levels, aphid colonies of the pest were raised on hot pepper plants in a screen house at Makerere University Agricultural Research Institute Kabanyoro (MUARIK), to generate individuals that were later introduced in known incremental numbers to hot pepper plants in exclusion cages. Yield loss data associated with specific initial pest densities were recorded. Results revealed that the yield loss caused by aphids increased with increase in infestation levels. The density of 2aphids/plant was found to be economically damaging and pesticide application should be initiated at a density of 1.5 aphids/plant to stop the increasing aphid density from reaching economic injury levels. From the study, it can be concluded that the fruit attacking pests in particular the fruit fly are the most damaging and so to ensure judicious use of pesticides, sprays should be initiated at fruiting stage; direct aphid damage can cause economic loss; and mixed cropping can be exploited in controlling aphids, thrips and whiteflies.
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    Prevalence and risk factors of Echinococcus granulosus infection in dogs in Moroto and Bukedea Districts in Uganda
    (Springer, 2015-10) Oba, Peter ; Ejobi, Francis ; Omadang, Leonard ; Chamai, Martin ; Okwi, Andrew Livex ; Othieno, Emmanuel ; Inangolet, Francis Olaki ; Ocaido, Michael
    A cross sectional study was conducted in Moroto and Bukedea districts of Uganda from May to September 2013 to determine the prevalence and risk factors of Echinococcus granulosus infection in dogs. Fresh dog faecal samples were collected, preserved in 70 % ethanol, and later screened for presence of taeniid eggs using zinc chloride floatation method. Positive samples were confirmed by a copro- PCR (polymerase chain reaction) for E. granulosus using NADH dehydrogenase sub-unit 1 gene (NADH1) as a target molecular marker. Structured questionnaires and focus group discussions were used to collect quantitative and qualitative data for risk factor identification. Study sub-counties were selected by simple random sampling. Overall apparent prevalence of taeniid infection in dogs of 14.9 % (39/261, confidence interval 10.6–19.2) in both districts was recorded using the faecal floatation test. The sensitivity of the faecal floatation test was found to be 78 % (25/32), while the specificity was 93%(215/229). Copro-PCR results revealed a true prevalence of 14.4% (9.91–19.0, 95 % CI) in dogs in Moroto district and 7.4 % (2.14–12.60, 95 % CI) in Bukedea district. The overall true prevalence of cystic echinococcosis (CE) was 12.2 % (8.70–15.76, 95 % CI) in both districts. The major risk factors identified using logistic regression were uncontrolled access of dogs to animal slaughter facilities, higher cattle herd sizes
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    Improving productivity of small East African goats using native browse species
    (Makerere University, 2015-12) Nampanzira, Dorothy
    Despite the socio economic importance of goats to the livelihoods of the people especially in the rural communities, their productivity in terms of meat production is low (UNDP, 2007). This has mainly been attributed to inadequate feeding and nutrition. Communities usually utilize feed resources that are locally available in their communities because they can get them at a cheap or no cost. There are several native feed resources present in the wild used in feeding goats in Buyende district; however, they have not been documented. Their spatial distribution, seasonal availability and accessibility are not known. As a result, the total quantity of the feed resources used in goat feeding in Buyende district is not known and therefore needs to be mapped out. Such baseline information is necessary in planning appropriate feeding strategies for the goats in the district in order to improve their productivity. In addition natural feed resources that exist in the wild are at a threat of extinction. This is due to the climate change and population pressure which has caused a change in the land uses (MEA, 2005). The grazing land is being converted to crop agriculture and settlement. This calls for conservation through domestication of the priority browses species to form a sustainable and accessible form of feed resource base. 5 The quality of the nutrition provided by the native feed resources to the goats is not known. The available information is from other regions and is largely limited to nutrient composition. As a result, their contribution to productivity has no scientific backup. There is a need to determine the quality of the feed in terms of degradability, intake, digestibility and N retention. Despite the fact that goat meat is more nutritionally acceptable to health conscious consumers compared to beef and mutton, the effect of supplementing diets based on these local browse species on weight gain and chemical composition, tenderness and fatty acid profiles of goat meat has not been studied as yet. Such information is necessary in the formulation of goat feed rations to be used in all production systems in order to improve the productivity of the goats in terms of quantity and quality. This study therefore, seeks to improve productivity of the goats in Uganda through improving nutrition quality using native browse species.
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    Prevalence of cystic echinococcosis in selected pastoral and agro-pastoral districts of Uganda
    (JSciMed Central, 2016-05-10) Othieno, Emmanuel ; Okwi, Andrew ; Mupere, Ezekiel ; Bimenya, Gabriel ; Zeyhle, Eberhard ; Oba, Peter ; Chamai, Martin ; Omadang, Leonard ; Inangolet, Francis O. ; Siefert, Ludwing ; Ejobi, Francis ; Ocaido, Michael
    A cross sectional ultrasound screening survey for human cystic echinococcosis (CE) was undertaken in the pastoral dist ricts of Moroto, Napak, Nakapiripirit and Amudat in Karamoja sub-region; and agro-pastoral communities of Teso region, in the districts of Kumi and Bukedea. Other areas of the survey included: Nakasongola in Central region and Kasese district in the Western region of Uganda. A total of 3,636 participants were screened and 67 cases (1.84%) had CE. The organ most affected was the liver 61.2% (41/67), followed by the kidney 17.9% (12/67), spleen 10.44% (7/67), omentum 8.95% 6/67) and lungs 1.5% (1/67). All districts screened had positive cases with the highest prevalence occurring in Napak (3.9%) and the lowest in Nakapiripirit (0.45%). The prevalence of CE (0.5±0.3%) in South Karamoja (Amudat and Nakapiripirit) was significantly lower (P<0.001, χ2= 18.98) than in Central Karamoja (Napak and Moroto, 3.32±1.3%). The prevalence of CE in Teso region was found to be 1.21±0.8%, Kasese 2.15±1.2 % and Nakasongola 2.7±1.3%. The prevalence in south Karamoja was lowest. Overall, there was no significant difference (P<0.05; χ2= 0.12) in prevalence between males (1.7%) and females (1.9%). However, in Karamoja females (2.2% CI: 0.8-3.6) were more likely to be infected (χ2= 16; P<0.05) with CE than males (0.9% CI: 0.1-1.7). Sixty four percent (n=43) of the cysts detected were viable
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    Evaluation of genoquick MTB dipstick assay for rapid detection of mycobacterium tuberculosis in sputum
    (Makerere University, 2017) Kaswabuli, Sylvia
    Tuberculosis, although a curable disease, continues to be one of the most important infectious causes of deaths worldwide (WHO, 2010). Globally, an estimated 1.4 million deaths occurred in 2011 as a result of infection with tuberculosis, one-fourth of the deaths were associated with HIV infections and most of it occurred in resource-limited settings (DeCock et al., 2013) where the burden of HIV infection is high. In Africa, tuberculosis (TB) is the leading cause of deaths among persons with HIV infections as the continent harbors 80% of the world HIV-TB cases (Corbett et al., 2003) and Uganda is ranked 20th of the TB high burden countries (WHO, 2006) The alarming increase in morbidity and mortality highlights the need to strengthen control measures. Diagnostic delay for TB is still considerable especially in developing countries which results in increased morbidity and prolonged transmission. Though it remains to be demonstrated, more sensitive, more rapid, and more patient-friendly diagnostic tools might have a significant impact on disease control by abbreviating diagnostic delay and reducing the period of transmission. Currently, TB diagnosis relies on traditional tests which include clinical and X-ray screening, conventional laboratory techniques including microscopy and culture, and of recent MTB/RIF GeneXpert, which are currently employed in countries like Uganda. Clinical and X-ray screening largely depend on the experience of the clinicians. Although sputum smear microscopy remains a laboratory diagnostic tool of choice for tuberculosis in resource limited settings (RLS) due to its simplicity, inexpensiveness and predictive power, it is affected by low sensitivity (60%) and its performance is often affected by poor equipment, trained manpower and quality assurance system in settings where resources are scarce (Parsons et al., 2011). Culture techniques are highly sensitive and specific, but the cost, technical complexity and time delay before results are available make culture not scalable for rapid detection and treatment of tuberculosis. Recent advances in TB have made it possible to use molecular technology like Xpert MTB/RIF (Cepheid) assay to detect mycobacterial DNA with great accuracy. This newly introduced molecular assay has shortened the turnaround time of the patient results and has the potential to detect Rifampicin resistance in the sample but the test is expensive and the costs can only be maintained by donations. This necessitates the need for more sensitive, specific and inexpensive point of care TB diagnostic tool(s). GenoQuick® MTB assay (Hain Lifescience, Nehrem, Germany), a test based on PCR and GenoQuick® technology has demonstrated its usefulness for the identification of other micro-organisms (Cohen-Bacrie et al., 2010) and has been adopted for the rapid diagnosis of TB (GQ-MTB) (Weizenegger, 2010). It enables the detection of Mycobacterium tuberculosis complex from decontaminated pulmonary and extra pulmonary clinical specimens. The principle of this new method involves sputum processing with 1% Nacetyl-l-cysteine (NALC), 4% sodium hydroxide and 2.9% sodium citrate (NALC-NaOH) in a biosafety cabinet to obtain a pellet, extraction of Mycobacterial DNA using Genolyse (HainLifescience, Nehrem, Germany) method followed by selective DNA amplification of IS6110 gene by polymerase chain reaction (PCR). Resultant single-stranded amplicons hybridize with specific MTBC probe that are included in the primer-nucleotide mix. This complex (MTBC probeamplicon) then binds selectively to the test band on the dipstick and is visualized by gold labeling on a lateral flow assay. When detectable adduct levels are reached, a golden color band is formed indicating a positive result. It is a fast, reliable and easy to use TB diagnostic method with the potential for becoming a point of care assay especially in high burden countries. Though highly promising, its diagnostic performance is yet to be evaluated especially in resource-limited settings like Uganda where TB is a burden. The present study aimed at evaluation of this assay using -20oC frozen NALC-NaOH processed sputum samples and directly on fresh sputum processed with a heat method that excludes a biosafety cabinet. The results of this study may have potential applicability in peripheral laboratory settings, with limited biosafety facilities.
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    Bacterial contamination of milk and antimicrobial resistance profiles of selected pathogens in Kiboga District
    (Makerere University, 2018) Mayanja, Raymond
    Milk when not properly handled during transportation and storage, favors growth of several microorganisms. Many factors exist in the cattle corridor of Uganda that suggest bacteria contamination of milk during transport to collection centres is an issue. A study was carried out to determine the bacteria contamination of milk along the transportation chain in Kiboga district using total plate counts (TPC) and characterize some of the isolated pathogenic bacteria using drug resistance/susceptibility patterns and molecular typing for resistance against vancomycin. Milk samples from 50 farms were collected at the farm and the same milk followed upto the collection centres and again collected. Bacteria contamination of raw milk was enumerated by TPC. The mean TPC was 6.16 X 106 cfu/ml in the farms and 6.62 X 107 cfu/ml in the collection centres with a minimum of 0 in the farms and 2300 cfu/ml in the collection centres. The maximum TCP was 48.6 X 106 cfu/ml in the farms and 1 X 109 cfu/ml in the collection centres. Generally, higher milk contamination was observed at the collection centres than in the farms (p = 0.000). Bacteria with the wide drug resistance patterns were Klebshiela spp and E.coli whereas those with the narrow resistance patterns were Staphylococcus aureus and Streptococcus spp. Pen G was the most inefficient drug with the following resistance patterns: Staphylococcus aureus 48%, CNS 91%, Klebsiella 96%, E.coli 100% and Streptococcus ssp 15%. The most effective drug was Ciprofloxacin with a susceptibility pattern among isolates as follows: Staphylococcus aureus 96%, CNS 61%, Klebsiella 100% and Streptococcus ssp 62%. The Van A gene associated with vancomycin resistance was detected in one Staphylococcus aureus isolate. No Van B gene was identified amongst the 15 vancomycin resistant isolates screened suggesting presence of other genetic markers for vancomycin resistance. In conclusion the bacteria contamination of milk in Kiboga district deteriorated during transportation due to the increase in bacterial load.