Theses and Dissertations Collection

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The evolutionary dynamics of genetic determinants of plasmodium falciparum resistance to sulfadoxine/pyrimethamine (sp) in South Eastern Tanzania
(Sokoine University of Agriculture, 2008) Malisa, Allen Lewis
This study reports a systematic follow up of genetic changes in the genes encoding the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes of Plasmodium falciparum, in isolates from three rural districts of South-eastern Tanzania. The enzymes are the target of antimalarial drug, sulphadoxine- pyrimethamine (SP). The population-wide analysis of resistance mutations in the dhfr and dhps genes was applied to examine the influence of different drug use policies and their potential for the selection for SP resistance. A total of 47 244 bloodspot filter paper samples were collected from all individuals of all ages in randomly selected households in a series of annual surveys conducted between 2000 - 2006. Twenty percent (9 662) of all samples were found positive for P. falciparum infection on microscopy and hence were used for the genetic studies. DNA was extracted from P. falciparum-positxvz samples and dhfr and dhps genes were amplified by a nested polymerase chain reaction (PCR) and resistance conferring point mutations determined. Size polymorphisms at three sets of microsatellite loci linked to dhfr and three other sets of unlinked microsatellite loci were analysed by PCR amplification and electrophoresis on an automated sequencer. The influence of National treatment policy on the parasite reservoir was profound. The change of first line therapy from CQ to SP brought about highly significant increase of the frequencies of dhfr triple and dhps double mutants. Artemisinin-based combination therapy (ACT- SP+Artesunate) in Rufiji had a small and non-significant impact on the frequency of dhps double and dhfr triple mutant alleles, but significantly disrupted their association. Z)A/r-l inked microsatellites revealed high diversity around the dhfr sensitive alleles and significantly reduced diversity around mutant dhfr alleles. The majority of triple mutant alleles had one flanking microsatellite haplotype which has previously been shown to be derived from Southeast Asia, while the double mutant alleles had multiple haplotypes which were independently derived. Distribution of major lineages indicates that there is extensive genetic exchange among the geographic regions. Unlinked microsatellites confirmed the extent of allele sharing among the regions and revealed a major trend for reduced transmission intensity, which was apparently independent of the ACT intervention.
Bioactivity of Synadenium glaucescens (pax) extracts on helminth eggs and larvae from wastewater stabilization ponds in Morogoro municipality,Tanzania
(Sokoine University of Agriculture, 2015) Hassan, S.
The increased use of wastewater and sludge from wastewater stabilization ponds in agriculture predisposes the consumers to the health risks. The objective of this study was to evaluate the bioactivity of Synadenium glaucescens (Pax) extracts on helminth eggs and larvae from wastewater and sludge in Morogoro. Helminth eggs and larvae were recovered using Bailenger and Baerman methods, and then quantified using McMaster techniques. Extracts (S. Glaucescens) used during hatchability and larvicidal bioassays were obtained using hot and cold solvent extraction. The effect of S. glaucescens extracts on hatchability and larvicidal was tested using in vitro and in vivo methods. One litre of wastewater was collected from anaerobic, facultative and maturation ponds. One hundred grams of sludge were sampled from the ponds and piles. Lethal concentration fifty (LC50) and inhibitory concentration fifty (IC50) were used to determine larvicidal and hatchability effects. The identified helminth eggs were from nematodes including Ascarid, Strongylid and Trichuris. Minimum amount of ≤ 1 e.p.l. were found in wastewater sampled from maturation ponds, same amount recommended by WHO guideline of 2006, and a maximum of 700 e.p.l. from anaerobic ponds. It was found that sludge samples contained a minimum of ≤ 1 e.p.g. from maturation ponds and maximum of 100 e.p.g. in anaerobic pond. The ethanol extracts of root bark and leaves were the most effective with IC50 19.34 and 39.56 μgml-1. The two extracts also demonstrated the highest LC50 of 19.41 and 30.19 μgml-1 respectively. The root bark extracts were more active than leaves extracts. This study demonstrated a high potential of using S. glaucescens extract in controlling helminths in wastewater and sludge.
Molecular diagnosis and characterisation of orf virus in symptomatic goats in coast and Dar es salaam regions, Tanzania
(Sokoine University of Agriculture, 2015) Mayenga, Charles
Orf virus (ORFV) is a member of the parapoxvirus genus that causes orf, a zoonotic and epitheliotropic highly contagious disease mainly affecting sheep, goats, wild ruminants and humans. In the present study, an outbreak of a disease in goats with clinical signs suggestive of orf in 11 flocks with a total of 259 goats was investigated between May and June 2015. Eight villages in districts of Bagamoyo (2), Ilala (1), Kinondoni (1) and Kisarawe (4) in Coast and Dar es Salaam regions of Tanzania were involved. The aim of the present study was to confirm ORFV involvement in diseased goats and to provide the genetic characteristics of ORFV. Upon visiting of goat flocks, a total of 72 goats presented orf-like clinical signs and 24 were reported to have died with similar clinical presentation. The presence of ORFV in oral swabs, scabs and skin scrapings was investigated by partial amplification of the ORFV RNA polymerase gene using Orf1 and Orf2 primers by polymerase chain reaction (PCR). A total of 16 out of 22 goats tested positive for ORFV upon PCR. Afterwards, molecular characterisation of ORFV was performed by amplification and nucleotide sequencing of the B2L gene encoding the major envelope protein. The results of nucleotide sequencing showed that orf was caused by closely related ORFV belonging to cluster I. ORFV were found to be genetically closely related to OV-SA00 (Accession number AY386264) strain of ORFV isolated in 2003 from scab material of a kid in United States of America and ORFV collected from a goat in Kyela, Tanzania in 2013. To our knowledge, this is the first report of the phylogenetic analysis of B2L gene of ORFV from goats in Tanzania. More studies are required to determine the extent of spread and genetic diversity of ORFV in livestock, wildlife and humans.
Polymorphisms of Kappa-casein and Beta-lactoglobulin genes and their association with milk fat and protein contents in Tanzanian goats
(Sokoine University of Agriculture, 2015) Kuwi, Salum Omari
This study was conducted to assess the polymorphisms of beta-lactoglobulin (-Lg) and kappa-casein (-Cn) genes and determine the effects of -Lg polymorphism on percentage of milk fat and protein contents in indigenous goat breeds found in Tanzania. Blood samples were collected from 22 - 24 unrelated animals for each breed. Genomic DNA was isolated from whole blood samples using Sepa gene extraction kit with some modifications. The -Lg and -Cn genes were amplified using Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) technique was used to determine variations at the -Lg and -Cn loci. The amplified products for -Lg and -Cn genes had the sizes of 426 and 459 bp, respectively. Digestion of β-Lg gene using SacII endonuclease restriction enzyme revealed the presence of two common alleles (A and B) in all goat populations. The frequency of B-allele (0.583) was higher than that of the A-allele (0.417) for β-Lg gene in indigenous goats, while exotic goats had higher frequency of A allele (0.688) than the B-allele (0.312). The frequencies of AA, AB and BB genotypes were, respectively, 0.250, 0.333 and 0.417 in Gogo goats, 0.318, 0.273 and 0.409 in Pare white, 0.417, 0.333 and 0.250 in Malya, 0.500, 0.375 and 0.125 in Saanen. The analysis of  -Cn gene variation in the four goat populations indicated that there were three alleles (A, B and C) at this locus. The observed allelic frequencies of A/B and C were 0.917 and 0.083 in Gogo white, 0.955 and 0.045 in Pare white, 1 and 0.00 in Malya, 0.875 and 0.125 in Saanen goats, respectively. The effects of β-Lg genotypes on milk fat and protein percentages were analyzed using a general linear model (GLM). The results show that there was no significant effects (P>0.05) of β-Lg alleles on milk fat and protein percentages.
Sero-surveillance, risk factors and molecular diagnosis of peste des petits ruminants virus in South Kivu, Democratic Republic of Congo
(Sokoine University of Agriculture, 2015) Ahadi, Bwihangane Birindwa
Peste des petits ruminants (PPR) is an acute viral disease of small ruminants caused by PPR virus (PPRV). For many years, the disease was mainly confined to West and Central Africa but has now spread southwards to previously PPR-free countries. The disease was first reported in Democratic Republic of Congo (DRC) in 2012. The disease causes high morbidities of up to 100 % and mortality rates between 50 and 90% in domestic small ruminants (goats and sheep) leading to the socio economic loss impact to the farmers. This study aimed to perform molecular diagnosis and sero-prevalence of PPR associated with transmission risk factors in unvaccinated sheep and goats from South-Kivu in province in DRC using respectively reverse- transcriptase polymerase chain reaction (RT-PCR), competitive enzyme-linked immunosorbent assay (cELISA) and a structured questionnaire. The results showed with cELISA an overall seroprevalence of 28.5% (n=319), out of which 11.3% and 32.7% seropositivity was found in sheep and goats, respectively. Peste des petits ruminants seroprevalence was higher in the territories that recorded high rainfall, 34.5% (n=142) in Shabunda and 29.4% (n=79) in Mwenga. In a total of 11 risk factors investigated four were found to be associated with PPR seroprevalence (p≤0.05). Among them we have animal’s age (OR: 9.34), grazing and farming system (6.28), territory geographic location (OR: 5.1) and the animal’s origin (OR: 0.5). Peste de petits ruminants seroprevalence was higher in small ruminants kept in communal grazing system (30.6%) and free ranging system (31.2%). Sheep and goats of >12 months had a significantly higher PPR seroprevalence (35.1%). As no PPRV RNA was detected in any of the blood collected using reverse transcriptase polymerase chain reaction (RT-PCR), we recommend further studies to be focused on molecular characterization and isolation of PPR virus.
Detection of Arenaviruses in rodents, shrews and elephant shrews from selected wildlife-human interfaces in Tanzania
(Sokoine University of Agriculture, 2015) Maganga, Ruth
The present study was conducted to investigate the presence of Arenaviruses from rodents, shrews and elephant shrews captured in selected wildlife-human interfaces in Tanzania. The study involved six sites with high potential for contact between wildlife and humans namely; Ruaha, Kilombero, Mtwara, Mbeya, Mbinga and Mikumi. A total of 121 animals comprising 111 rodents, 3 shrews and 7 elephant shrews were screened for Arenaviruses using conventional Polymerase Chain Reaction (PCR). The genetic relatedness of Arenaviruses was evaluated by conducting phylogenetic analysis of partial sequences of the S gene. The association between age and sex with the presence of Arenaviruses was assessed. Of the 121 animals, 7 (5.8 %) were shedding Arenavirus. All positive animal samples were obtained from the Ruaha site at crop raiding and peridomestic interfaces. Eighty six percent of the infected animals were Mastomys sp. and 14 % were Arvicanthis sp. Age and sex of the animals were not significantly associated with occurrence of Arenaviruses in rodents, shrews and elephant shrews (P > 0.05). Additionally, Arenavirus detection in fecal specimens was not significantly different from the detection in oropharyngeal specimens (P > 0.05), clearly indicating that both specimens are useful for Arenavirus testing. Phylogenetic analysis showed that isolates obtained from this study were related to the Old World Arenaviruses, and include strains of Morogoro virus and a novel strain of Arenavirus. In conclusion, the present study has confirmed the presence of Arenaviruses closely related to other known Old World Arenaviruses in the Ruaha ecosystem.
Molecular epidemiology and antimicrobial resistance of thermophilic campylobacter infections in humans and animals in Tanzania
(Sokoine University of Agriculture, 2015) Komba, Erick Vitus Gabriel
Members of the genus Campylobacter are known to cause more cases of human gastrointestinal illness than any other bacterium worldwide. The organisms exist as normal flora in the intestinal tracts of domestic and wild animals, more so in avian species. Humans acquire Campylobacter infections from contaminated animal products, particularly poultry meat, either directly or through cross-contamination of other food products. Human infections are mostly attributed to Campylobacter jejuni and C. coli, the former causing a larger proportion (85-90%) of all cases reported. In addition to infections, campylobacteriosis is also associated with the emerging threat of antimicrobial resistance as evidenced in isolates derived from different sources. An accurate picture of the epidemiology of infections caused by Campylobacter and other aetiological agents is lacking in developing countries due to the absence of regular surveillance programmes. Consequently the present study was conducted in Morogoro Municipality, Eastern Tanzania, to determine the molecular epidemiology and antimicrobial resistance of thermophilic Campylobacter isolates from humans and animals. Specific objectives were; 1) To establish the prevalence of thermophilic Campylobacter infections in humans and animals, 2) To determine the genetic relatedness of chicken and human derived thermophilic Campylobacter isolates using DNA-based typing methods, 3) To evaluate the antimicrobial resistance patterns in thermophilic Campylobacter isolates derived from humans and animals; and 4) To identify risk factors for thermophilic Campylobacter infections in humans. Stool samples were collected from 1195 human subjects; and fecal samples from 1511 farm animals, 466 laboratory animals and 112 wild birds (Indian house crows). Farm animals constituted chickens (n=1267), cattle (n=98), goats (n=81), sheep (n=57), horses (n=5) and camels (n=3); whereas laboratory animals were composed of guinea pigs (Cavia porcelllus, n= 30), mice (Mus musculus, n=160), rabbits (Oryctulagusiii cuniculus, n=34) and rats (Rattus rattus, n=242). The Cape Town protocol was used for isolation of thermophilic Campylobacter from stool and fecal samples. Campylobacter isolates were identified by phenotypic and molecular techniques. The isolates were tested for resistance against several antimicrobial agents using the disc diffusion method. Risk factors for human infections with thermophilic Campylobacter were determined in an unmatched case control study. Selected human and chicken derived Campylobacter jejuni isolates were genotyped using flagellin A gene sequencing. In humans the prevalence of thermophilic Campylobacter was 11.4% (n=1195). Symptomatic (12.9%) and young individuals (16.7%) were more infected than asymptomatic (6.7%) and adults (10%), respectively. Most (84.6%) of the isolates were C. jejuni and the remaining were C. coli; and the difference was statistically significant at p≤0.05. Isolates had highest resistance (95.6%) for colistin sulphate and lowest for ciprofloxacin (22.1%). Proportions of resistant isolates for other antibiotics (azithromycin, erythromycin, tetracycline, cephalethin, gentamycin, nalidixic acid, ampicillin, amoxycillin, norfloxacin and chloramphenicol) ranged from 44.1% to 89%. Human infections with thermophilic Campylobacter were associated with young age; and consumption of chicken meat, barbecue and pre-prepared salad. In avians, thermophilic Campylobacter spp. were isolated from 44.0% and 20.5% of the sampled chickens and crows respectively. The majority of isolates from both chickens (87.6%) and crows (56.5%) were C. jejuni and the remaining were C. coli. The observed difference in proportions of C. jejuni and C. coli isolates was statistically significant (p≤0.05) in chickens but not in house crows. Chicken isolates had highest resistance to Colistin sulphate whereas crow isolates showed highest resistance to azithromycin and erythromycin. Lowest resistance was observed for gentamycin and ciprofloxacin for crow and chicken isolates respectively. Among chicken isolates significantly high proportions of C. coli were resistant to gentamycin, cephalothin, tetracycline, colistin sulphate and chloramphenical. On the other hand a high proportion C. jejuni isolates were resistant toiv nalidixic acid. Crow derived C. jejuni had significantly higher resistance to nalidixic acid, cephalothin and ciprofloxacin than C. coli isolates from the same hosts. Among farm animals thermophilic Campylobacter were detected from 18 (31.6%) sheep and 3/5 (60%) of horses. Of the isolates 12 (57%) were C. jejuni; the remaining (43%) were C. coli. Of the laboratory animals 8 (26.7%) guinea pigs and 3 (1.2%) rats were colonized with Campylobacter. Four isolates from the guinea pigs were C. jejuni and the other 4 were C. coli. From the rats two isolates were C. jejuni and one was C. coli. The isolates showed high levels of antimicrobial resistance to erythromycin, norfloxacin colistin sulphate and nalidixic acid in ascending order; whereas low levels of resistance were observed for ciprofloxacin and gentamycin. Out of 55 sequenced isolates obtained from sporadic cases of human illness and different categories of chickens, nine different flaA types (7, 36, 41, 51, 61, 62, 64, 105 and 111) were detected. Both C. jejuni isolates from humans and chickens displayed a high degree of genetic diversity thereby suggesting weak clonality among the tested isolates. Genetic relatedness of some isolates from human and avian sources was however evident as on phylogenetic analysis some clusters contained both human and chicken C. jejuni isolates. The work contained in this thesis contributes significantly to the limited, available information on epidemiology and antimicrobial resistance of human and animal Campylobacter infections in Tanzania. For the first time the occurrence of Campylobacter infections in laboratory animals, antimicrobial resistance of human derived Campylobacter isolates, risk factors for human Campylobacter infections; and the population structure and relatedness of Campylobacter jejuni isolates from humans and chickens in the country are provided. The observed clusters containing isolates from human and avian sources confirm interspecies transmission of this zoonotic pathogen. Information on antimicrobial resistance of Campylobacter isolates derived from avian species in the country is also complemented. Control measures for colonization of animals and occurrence of infections in humans with this particular bacterium species arev warranted. Similarly strategies to stem emergency and spread of antimicrobial resistant Campylobacter strains should be put in place.
Molecular epidemiology of Leptospira species among Agropastoral communities living in Katavi-Rukwa ecosystem, Tanzania
(Sokoine University of Agriculture, 2015) Muller, Shabani Kililwa
Leptospirosis is an emerging zoonotic infectious disease which affects humans and animals worldwide as it causes febrile illness in humans. The disease has been reported in a number of human-livestock-wildlife interfaces of Northern and Eastern Tanzania. Very little is known of many zoonotic disease conditions in the research naive areas of Western and Southern Tanzania. This study aimed at detecting the prevalence of Leptospira species among agro-pastoralists at the human-animal interface areas of Katavi-Rukwa ecosystem. Microscopic agglutination test (MAT) was used to detect antibody against six Leptospira antigens including local serogroups Icterohaemorrhagiae, Ballum, Grippotyphosa, Sejroe and reference serogroups Hebdomadis and Lora. Samples with MAT titers ≥ 1:160 were scored as positive while MAT titers between 1:20 and 1:80 were scored as exposed to Leptospira and absence of agglutination titers was scored as negative. Of the 267 samples tested 80 (30%) were positive, 57 (21.3%) were negative and 130 (4 8.6%) were exposed to leptospiral infection. Infection rate in adults was higher 51 (63.75%) compared to children 29 (36.25%), P<0.05. Circulating serogroups were; Hardjo (15.7%); Icterohaemorrhagiae (8.98%), Grippotyphosa (4.87%), Hebdomadis (3.37%), Australis (1.49%) and Ballum (1.12%). Samples that were positive or scored as exposed by MAT were further tested using polymerase chain reaction (PCR) targeting 16S ribosomal gene. Pathogenic Leptospira was detected in 33 (15.5%) out of 212 while no saprophytic Leptospira species was detected. Sequencing alignment based on 16S ribosomal gene suggested Leptospira interrogans, kirshinei and uncultured Leptospira clone species as circulating species among agro-pastoralists of Katavi-Rukwa ecosystem. These findings suggest that in the Katavi leptospirosis in man is likely acquired from environment, probably by indirect contact with contaminated water or soil. This study also revealed that serological diagnosis of leptospirosis should be considered in the diagnosis on non- malarial febrile illness in agro-pastoralists living in Katavi-Rukwa ecosystem, Tanzania.
Detection of Arenaviruses in rodents, shrews and elephant shrews from selected wildlife-human interfaces in Tanzania
(Sokoine University of Agriculture, 2015) Maganga, R.
The present study was conducted to investigate the presence of Arenaviruses from rodents, shrews and elephant shrews captured in selected wildlife-human interfaces in Tanzania. The study involved six sites with high potential for contact between wildlife and humans namely; Ruaha, Kilombero, Mtwara, Mbeya, Mbinga and Mikumi. A total of 121 animals comprising 111 rodents, 3 shrews and 7 elephant shrews were screened for Arenaviruses using conventional Polymerase Chain Reaction (PCR). The genetic relatedness of Arenaviruses was evaluated by conducting phylogenetic analysis of partial sequences of the S gene. The association between age and sex with the presence of Arenaviruses was assessed. Of the 121 animals, 7 (5.8 %) were shedding Arenavirus. All positive animal samples were obtained from the Ruaha site at crop raiding and peridomestic interfaces. Eighty six percent of the infected animals were Mastomys sp. and 14 % were Arvicanthis sp. Age and sex of the animals were not significantly associated with occurrence of Arenaviruses in rodents, shrews and elephant shrews (P > 0.05). Additionally, Arenavirus detection in fecal specimens was not significantly different from the detection in oropharyngeal specimens (P > 0.05), clearly indicating that both specimens are useful for Arenavirus testing. Phylogenetic analysis showed that isolates obtained from this study were related to the Old World Arenaviruses, and include strains of Morogoro virus and a novel strain of Arenavirus. In conclusion, the present study has confirmed the presence of Arenaviruses closely related to other known Old World Arenaviruses in the Ruaha ecosystem.
Assessment of genetic diversity of maize landraces in Tanzania using Random Amplified Polymorhic DNA markers
(Sokoine University of Agriculture, 2016) Asigbee, T. W.
The knowledge and comprehension of the genetic variation of maize (Zea mays L.) landraces is pivotal for the implementation of measures to address conservation and improvement. The purpose of this study was to assess the genetic diversity and relationship among selected maize genotypes in Republic of Tanzania by screening twenty Random Amplified Polymorphic DNA molecular markers.DNA was extracted from 160 maize genotypes and PCR was conducted using twelve informative primers. Amplification revealed 104 polymorphic bands with an average of 8.67 polymorphic fragments per primer. The number of amplified fragments ranged from 7 (OPP-02) to 10 (OPK-08), with the amplicon sizes ranging from 75 to 2000 base pairs. The polymorphic information content (PIC) ranged from 0.7487 to 0.954 with an average of 0.8647 and gene diversity value ranged from 0.7531 to 0.9577 with an average of 0.8698.The dendrogram drawn based on Neighbour- Joining method revealed the diversity and genetic relatedness among the landraces in the various clusters but did not reflect the geographical locations of the studied maize genotypes. This might be attributed to the high gene flow in the various study locations.The analysis of the RAPD molecular markers revealed a high genetic diversity among the maize landraces and proved to be a practical method for assessing polymorphism in maize cultivars. These findings will be useful to establish and improve the current germplasm collection of landraces and help maximize the utility of maize genetic resources.
Adaptation of reverse transcription loop- mediated isothermal amplification for field diagnosis of foot-and- mouth disease in Tanzania
(Sokoine University of Agriculture, 2016) Kandusi, Sengiyumva Emmanuel
Foot-and-mouth disease (FMD) is a highly contagious viral vesicular disease of cloven hoofed animals and poses major constraints to international trade in livestock production. Methods available for detection of FMD virus (FMDV) require specialized laboratory facilities and equipment. In this study, targeted laboratory- based experiments studies were conducted using reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection and serotyping of FMDV under field conditions. Pan-serotypic RT-LAMP utilizing labeled and unlabeled primers was used for detection of the virus. Serotype-specific primers for FMDV serotypes A and O were used to type the positive samples using RT-LAMP. Amplification was observed in real-time for unlabeled primers and by molecular lateral flow devices for labeled primers. Also, gel electrophoresis was used for examination of deoxyribonucleic acid (DNA) bands. A total of 35 samples (n = 35) were examined using RT-LAMP. Of these, 40% (n=14) were positive from different regions in Tanzania. The positive samples were from Iringa with 29% (n=4), Morogoro with 14.2% (n=2), as well Kilimanjaro, Mara, Tanga, Tabora, Mtwara, Kagera Dar es Salaam and Mwanza with 7.1% (n=1) each. All the pan- serotypic RT-LAMP positive samples revealed time for positivity ranging from 12-30 minutes. These findings indicate that the standardized RT-LAMP assay reported in this study can be used for field detection of FMDV in suspected FMD outbreaks in Tanzania. These findings suggest a potential use of serotype-specific RT-LAMP for typing FMDV field strains.
Determination of the level of expression of OsCIPK15 salt responsive gene in selected Tanzanian Rice Landraces
(Sokoine University of Agriculture, 2016) Kamanga, Naomi Olga
This was an experiment to determine the presence and level of expression of OsCIPK15 salt responsive gene in Tanzanian rice breeders’ lines. Abiotic stress is one of the factors affecting rice cultivation in Tanzania. The calcineurin B- like protein interacting protein kinases (OsCIPKs) responsive genes have been observed to express in abiotic stress. The calcineurin B- like protein interacting protein kinases-15 (OsCIPK15) salt responsive gene, which is usually a silent gene expresses in saline soils which is abiotic factor affecting yield. In this experiment eighty-four breeders’ lines were used for the study. The lines were grown in sterilized sandy soil and grown for two weeks in the greenhouse. After this period the two week old seedlings were uprooted and the roots submerged in saline solution of 200mM concentration for forty-eight hours. Leaf samples were collected exactly twenty-four hours, twenty-nine and forty hours and stored at -80˚C The samples were thereafter analysed using quantitative real-time polymerase chain reaction (QRT-PCR). Of the eighty-four breeders’ lines twenty-two gave a quantifiable analysis using the Livak delta analysis. Of the lines CSR 27, TXMS 1-2, TXM 18-1 and TXM 27 were the most tolerant and expressed the gene highly and TXM 13-2-3, GIZA 179, TXMS 14 and TXM 13-2-1 were the least. This experiment proves that Tanzanian breeders’ rice have inherent ability to tolerate abiotic stress, such as salinity and the lines studied can be used in breeding programs to develop rice salt tolerant varieties to be cultivated in susceptible areas in order to provide profitable yield for paddy growing rice farmers.
Responses of crossbred calves to theileria parva infection following targeted stimulation with Toll-like receptor 7 (TLR7) agonist
(Sokoine University of Agriculture, 2016) Sarfo, Kelly Owusu
Control of East Coast fever (ECF) depends on the use of acaricides against ticks, chemotherapy and vaccination. ECF vaccination is based on an infection and treatment method (ITM), which induces life-long immunity if cattle are exposed to mild tick infestation. However, ECF vaccination has several limitations, such as high cost of simultaneously used antibiotics. The primary objective of this study was to determine whether Toll-like receptor 7(TLR7) agonist could effectively induce strong immunity in calves prior to ECF vaccination and replace the use of long acting tetracycline. Twenty crossbred calves were split into 4 groups; adjuvant (N=10), vaccinated (N=4), infected (N=4) and control (N=2). Stimulation of calves with TLR7 agonist induced a strong innate response in terms of a rapid rise in temperature, skin inflammatory response and pronounced swelling of the lymph node in calves. TLR7-priming induced a significant impact on the response of the calves to subsequent T. parva infection. In comparison to the infected group, lymphocyte counts were higher in calves which received the adjuvant. Furthermore, the combined effect of TLR7 agonist stimulation and T. parva parasite challenge induced a high level of IFN-gamma response almost similar to the response shown by ECF vaccinated calves. Similarly, the adjuvant group attained higher antibody level earlier than the infected control calves. Based on the clinical signs observed from day 14 onwards, calves which received TLR7 agonist developed milder disease signs compared to the infected control calves, which were all treated against ECF on day 18. Only one calf out of ten TLR7-primed calves developed clinical disease signifying a potential adjuvant role of the agonist in ECF vaccination. Possibly the adjuvant acted in this mechanism. This study has demonstrated a practical application TLR7 agonist in ECF vaccination, without simultaneous use of antibiotics.
Diversity of extended-spectrum beta-lactamase genes in avian pathogenic Escherichia Coliin scavenging local chicken in Morogoro Municipality, Tanzania
(Sokoine University of Agriculture, 2016) Armah, Emmanuel Odartei
The poultry industry, especially chicken production has in recent times faced a major set-back due to devastating effects of Extended Spectrum Beta-Lactases (ESBL) producing organisms.This research aimed at investigating the diversity of ESBL genes in avian pathogenic Escherichia coli(APEC) among scavenging local chickens. A total of 400 cloacal and oropharyngeal swabs were obtained, out of which 192 Escherichia coli were isolated. By use of virulence factor profiling, these 192 samples were screened for the presence of 16 virulence factors by multiplex PCR. All 192 samples harbored at least one of the 16 virulence genes and 19 of them carried at least four, making them APEC. The virulence traits ibeA, iss, traT and chuA were observed to lead the chart with percentages of 84.21, 78.95, 63.16 and and 52.63respectively. In the pathogenesis of APEC, Iron acquisition, serum resistance, toxins and invasins were found to be very significant (P<0.05). The antimicrobial profiles of these APEC strains were determined by Kirby-Bauerdisc diffusion test using 10 antimicrobials. These include:augmentin (30μg), imipenem (10μg), cephalothin (30μg), cefotaxime (30μg), ceftazadime (30μg), ceftriaxone (30μg), nalidixic acid (30μg), ciprofloxacin (5μg), gentamycin (10μg) and trimethroprim-sulfamethoxazole(25μg). All APEC strains were found to be resistant to at least one of these drugs, with 10.52% of them being multi-drug resistant. By double-disc synergy test, eight of the APEC isolates were found to be ESBL producers. They were screened for the presence of beta-lactamase genes and the following were present: blaTEM-100%, blaOXA-1 -75.0%, blaCMY-2 62.5%, CTX-M group III (CTX-M-8)-50%, CTX-Mgroup IV (CTX-M -9)-37.5%, CTX-M group I(CTX-M -1,and-15) -12.5% and blaSHV-12.5%. Occurrence of virulence strains of APEC and ESBLs genes are also alarming
Association of Betaine Aldehyde Dehydrogenase 2.1 (Badh2.1) Gene Allele with aroma in popular traditional rice varieties in Tanzania
(Sokoine University of Agriculture, 2016) Moshi, W. E.
Aromatic rice is highly cherished in many countries of the world and commands premium prices at all levels of the global rice trade. The presence of aroma in aromatic rice is controlled by betaine aldehyde dehydrogenase 2.1 (BADH2.1) gene allele which results from an eight base pair deletion and three single nucleotide polymorphisims (SNPs) in exon seven of betaine aldehyde dehydrogenase 2 (BADH2) gene. This mutation is responsible for the introduction of premature stop codon which produce a truncated protein, this results in loss of function of the enzyme betaine aldehyde dehdrogenase 2 (BADH2) leading to accumulation of substrate (main aroma compound) 2-acetyl 1-pyrroline (2AP) in aromatic rice varieties. In this study, the association between BADH2.1 aromatic allele and aroma in Tanzanian rice varieties was investigated. Leaf and grain aromatic tests for aroma evaluation and screening for BADH2.1 gene allele using allele specific amplification (ASA) marker were conducted in 160 popular traditional rice landraces from different geographical regions of Tanzania. Of the 160 landraces genotyped and phenotyped; 95 varieties were classified as aromatic by the presence of aroma in both leaf and grain aromatic tests, most of these (91.6 %) carried BADH2.1 gene allele. Evidence from sequencing of BADH2/BADH2.1 alleles confirmed the association of BADH2.1 gene allele with aroma in aromatic rice landraces as it was shown that all aromatic genotypes had eight base pair deletion and three SNPs in exon seven of BADH2 locus. This suggests that BADH2.1 gene allele is the main aroma allele in most of the Tanzanian aromatic rice varieties. Phylogenetic analysis of BADH2/BADH2.1 nucleotide sequences, showed a large amount of genetic variability (39.41-100 % nucleotide sequence identity) among the varieties studied. These findings will contribute siginificantly in planning for effective rice breeding strategies especially in selection of appropriate parental materials for developing high yielding aromatic rice varieties in the country.
Genetic and antigenic characterization of foot and mouth disease virus strains isolated in 2011 and 2015 in Ngamiland, Botswana
(Sokoine University of Agriculture, 2016) Seoke, L.
Foot-and-mouth disease (FMD) is a highly contagious disease of cloven hooved animals that continues to occur in Ngamiland District of Botswana although stringent disease control measures have been put in place. This may be due to irrelevance of currently used vaccine strains. In the present study, genetic and antigenic characteristics of SAT2 viruses isolated from outbreaks which occurred in 2011 and 2015 in Ngamiland districtwere examinedin order to determine any mutational changes of the FMD viruses circulating in that area. The antigenic relationships between the outbreak strains and SAT2 vaccine strains currently used were also determined. Tissue samples collected were subjected to sequencing of the VP1 gene, phylogenetic analysis and vaccine matching with the two SAT2 vaccines strains currently in use in the same region; SAT251 and SAT2035. There was almost 100% amino acid sequence similarity within both outbreaks while minimal mutations (90% sequence similarity) occurred between the outbreaks. Phylogenetic analysis revealed that both outbreaks were caused by genetically similar viruses that belong to SAT2 topotype III. The newer vaccine strain, SAT2035 clustered with the field virus isolates on the phylogenetic tree indicating that it also belongs to the same topotype. However, the older vaccine strain, SAT251, was shown to belong to topotype II. Amino acid variation analysis revealed that mutations occurred post the 2011 outbreak but did not impact on the antigenicity of the field isolates although majority of variability occurred at known FMDV antigenic sites. This is confirmed by r1values obtained against both vaccine strains. The findings are evidence that the vaccines provide satisfactory immunity and are still relevant to confer protection against circulating field strains in the area because minimal mutationsoccurred intra- and inter-outbreaks. Recurrence of the disease is probably due to low vaccination coverage and this should be improved.
Detection and characterization of mosquito-borne viruses circulating in mosquitoes of Morogoro Municipality, Tanzania
(Sokoine University of Agriculture, 2016) Ahedor, Believe
Mosquito-borne viruses primarily infect animals and humans causing significant public and veterinary health threat. Transmitted by hematophagous mosquitoes, dengue virus and Rift Valley fever virus are the most common outbreaks that occurred in parts of Tanzania especially in malaria endemic areas. Several studies reported the presence of the viruses in circulation in animals and humans. Therefore, this study sought to investigate the diversity of mosquito-borne virus vectors and to assess the risk of mosquito-borne viral transmission in Morogoro municipality. Molecular detection of these viruses was carried out on Aedes aegypti using reverse transcription polymerase chain reaction (RT-PCR). A total of 7649 mosquitoes comprising of 7224 adults and 424 larvae belonging to five genera (Aedes, Anopheles, Culex, Eretmapodites, Mansonia) and 14 species were collected. The predominant specie was Culex quinquefasciatus 53.9% (n=3891) and Aedes aegypti 44.2% (n=3192), most of the species 43.7% (n= 3156) were collected in Mbuyuni, 22% (n=1587) from Kiwanja cha Ndege, 19.2% (n=1387) from Mwembesongo and Mazimbu and Kilakala with 7.6% (n=549 and 545) respectively. About 70% of the Aedes spp were collected from used car tyres - the major breeding sites. The mosquitoes’ 18S ribosomal ribonucleic acid (rRNA) and viral RNA were successfully amplified, but no specific viruses were detected. However, for the mosquito pools positive for flavivirus, by sequencing the generated PCR products, it was found that there could be false positives due to non-specific amplification of mosquito ribosomal RNA or amplification of arboviral-like sequences integrated into the Ae. Aegypti genome. Nonetheless, this result provides an insight into the abundance and distribution of potential vectors in these wards. The close proximity of these vectors to humans poses high risk of virus transmission in the municipality and calls for rational vector control measures
Molecular diversity of DNA-B component of east African cassava mosaic viruses in Tanzania
(2017) Lupembe, M. D.
Begomoviruses are whitefly-transmitted viruses belonging to the family Geminiviridae presenting most devastating threat to food security. This study was initiated to asses cassava mosaic disease (CMD) severity and genetic diversity of DNA-B component of East African cassava mosaic viruses (CMD) that infect cassava plants from main cassava growing regions in Tanzania. The study involved field survey, rolling cycle amplification (RCA) followed by next generation sequencing (NGS). The symptoms of CMD severity in the areas where samples were collected ranged from moderate (2.82) to high (3.95) using a 1-5 (CMD symptoms severity score scale). Nucleotide sequence identity of 39 isolates identified in this study and that of 32 sequences retrieved from database ranged from 30% to 100%. Current study has reported the presence of EACMMV-Malawi (Accession number JX658684) from isolate TZ_KIB1 shared 98% nts identity and 93% to 98% nt identity with other isolates that were found in the same cluster (branch) with 2753 nts sequence length. Moreover, the presence of four isolates TZ_MBE, TZ_SUM, TZ_KIR and TZ_KAS from southern zone has also been described, shared 97% to 100% nts identity with EACMCV-TZ1 (Accession number AF112355) and 100% nts identity with other isolates from the study. Also, African Cassava Mosaic Virus (ACMV) from isolate TZ_TAR1 with 100% nucleotides (nts) identity to ACMV [(Nigeria /Ogo) Accession Number AJ427911] and 2718 nts sequence length from lake zone of Tanzania has been identified. Moreover, in this study isolates TZ_TAR3, TZ_ROR1, TZ_MIS, TZ_HAN and TZ_KIB1 showed recombination associated within themselves as well as with other isolates identified in the previous studies. In conclusion, the current study showed a greater genetic diversity of begomoviruses DNA-B being more diverse than previously thought that could be linked to geographical area and high rate of recombination.
The effect of cold storage and cooking procedures on the levels of oxytetracycline residues in beef from Dodoma Region, Tanzania
(Sokoine University of Agriculture, 2018) Mgonja, F. R.
Worldwide, there is an increased use of antimicrobial drugs due to occurrence of diseases of human and animals. The general objective was to study the effect of cooking procedures and cold storage on the levels of Oxytetracycline (OTC) residues in beef in Tanzania. The study used a cross-sectional research design whereby both quantitative and qualitative data were collected from Dodoma region, Tanzania. The household survey was conducted to assess knowledge, attitude and practice on beef consumption among 254 residents. The results show that community based health education and promotion of proper antimicrobial use in animals and preventing drug residues is highly recommended to this population. Beef samples were also analyzed by using High Performance Liquid Chromatography Mass-Spectrometry (HPLC-MS). The quantitative data were analyzed using the IBM Statistical Package for Social Science (SPSS) software version 20 and Epi info version 7. A simple and sensitive method for the detection of OTC levels in ready-to- eat beef by HPLC-MS was modified and validated and used for beef analysis in this study. The advantages of the modified method were cleaning by Supelclean ENVI-carb active coal is cheaper compared to solid phase extraction and samples drying using a stream of liquid nitrogen is cheaper and more than six samples can be dried at a time. For the raw beef, the results indicate that the mean concentration level of OTC was very low (0.69 ± 0.09 ng/g). The boiled and barbecued beef, the mean concentration was 69.4 ±41.93 ng/g and 69.40±38.91 ng/g, respectively. The results indicate that one should not count on heat- treatment to eliminate residues of OTC from beef. The effect of the cold storage on the concentration of OTC residues in beef stored at -20 °C for 60 and 120 days showed that the mean concentration of OTC residues before freezing was 191.71 ± 90.21 ng/g. The mean concentration of OTC after freezing at -20 oC for 60 and 120 days were 166.40 ± 86.49 ng/g and 133.50 ± 83.24 ng/g respectively. These results revealed a significant (p<0.05) reduction of OTC residues of 30% after 60 days and 65% after 120 days of freezing at -20 °C.
Cyanide levels in raw sweet cassava varieties and people’s perception on cyanide poisoning in Kagera and Morogoro regions of Tanzania
(Sokoine University of Agriculture, 2018) Mushumbusi, C. B.
Cassava (Manihot esculenta Crantz), an edible crop that is renowned out of its growth advantages over other crops, carries cyanide which is potentially poisonous to humans. The threat is intensified by human habit of consuming raw cassava tubers whose toxicological status is not established. Cases and indicators of cyanide poisoning due to cassava consumption are evident in Tanzania, particularly in Kagera and Morogoro regions. This study was launched to quantify cyanide in tubers of sweet cassava varieties grown in Kagera and Morogoro regions and thereafter to assess if it is safe for humans to consume raw tubers of sweet cassava varieties grown in the study area. Another objective was to assess whether the habit of consuming raw cassava tubers was out of people’s lack of awareness about cyanide and its poisoning effects or negligence. The study employed cross-sectional research design to collect participants’ responses on cyanide and its associated poisoning effects and to determine cyanide levels in sweet cassava tubers by using alkaline titration method. Sixty six tubers of 12 sweet cassava varieties from the study area were analyzed and 386 participants were involved in the study. The study findings showed that cyanide levels in the raw sweet cassava tubers were above the internationally accepted level in human consumables (10 mg/kg) and thus unfit for human consumption in their raw state. Some sweet varieties were found to be wrongly classified as sweet because their cyanide content was above 50 mg/kg. The inconsistency of cyanide levels in tubers of similar variety showed that a variety can exist in both sweet and bitter forms depending on environmental factors, making the categorization of varieties into sweet and bitter varieties misleading. Furthermore, the habit of consuming raw cassava tubers was found to be mostly cultivated by people’s lack of awareness with regard to the presence of cyanide in cassava tubers (86%) and on cyanide poisoning effects (81%) respectively. It was also found out that the slippery tissue and the inner tissue of the cassava parenchyma differ significantly in cyanide content so that the habit of scratching off the slippery tissue contributes to reduction of cyanide in cassava tubers. This research work recommends that the public should be sensitized on the issue of cyanide in cassava, the poisoning effects it has to human health as well as ways of identifying and dealing with the poison contained in cassava tubers prior to human consumption. Apart from inventing simple, inexpensive and efficient cyanide quantifying devices, genetically modified cassava varieties need be produced and disseminated in which the gene for cyanide expression is either masked or removed as an attempt to protect people against cyanide poisoning.

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