PHD-Department of Biochemistry and Biotechnology

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Population genetics and taxonomy of important medicinal tree species of the genus Warburgia
(Kenyatta University, 2007-07) Muchugi, Alice Njeri
The genus Warburgia (Canellaceae) has only four species (W. elongata Verdc., W. salutaris (Berto; f). Chiov, W. stuhlmannii Engl. and W ugandensis Sprague, which occur in east and central Africa. These species are of valuable medicinal importance to the local communities where they occur and have also gained interest from international herbal markets. The taxonomy of the species in this genus is conflicting with some authorities referring to the distinct species as con specific or synonyms. Genetic diversity present in wild populations of these species is under great threat due to unsustainable harvesting for medicines and indiscriminate felling trees and therefore there is an urgent need for conservation of these species. On farm planting is one of the best alternatives for ensuring the survival of the species and sustainable harvesting for their medicinal products. This research aimed to resolve the taxonomic confusion using molecular markers and to determine the genetic relationships within and among populations of the species in the genus Warburgia in order to guide strategies for their conservation and sustainable utilisation. Ecological survey showed reduced populations sizes of Warburgia species, which has being caused by land clearance for farming and settlement, timber, charcoal and fuelwood harvesting. The study showed a discontinuous tree distribution where young plants were not replacing mature trees coming to the end of their reproductive life. Significant differences were found in fruit and seed size between W ugandensis and W stuhlmannii. The chromosome number in W ugandensis and W stuhlmannii was found to be 2n=22 for both species, which implied that the species have conserved the basic chromosome number in the genus despite adapting to divergent ecological zones. The genetic relationships among Warburgia species were determined using the AFLP technique. Genomic DNA for the AFLP analysis was obtained through the CTAB method, which proved to be the best in efficiency and cost among the tested methods. Analysis of molecular variance (AMOV A) on data obtained revealed most genetic variation being among individuals within populations (63%, P< 0.0001), but variation among populations (37%, P< 0.0001) was highly significant as well. High genetic diversity was found within the studied populations. A phenetic tree and constrained ordination analysis illustrating the relationship among populations of Warburgia species showed a clear distinction among Warburgia species. A populations' subgroup of W ugandensis from Uganda and western Kenya clustered away from the rest of W ugandensis implying genetic distinctiveness while the coastal species (W salutaris and W stulhmannii) showed little genetic differentiation. The utility of AFLP and RAPD techniques to resolve intraspecies genetic variation in W. ugandensis populations was assessed and the two techniques showed high correlation (r=78, P< 0.001). However, AFLP technique had a higher marker index (24.96) than RAPD analysis (19.70). Assessment of type of mating system in W. ugandensis using the AFLP markers showed the species to be predominantly outcrossing (88.9%) with a low level of selfing (11.1 %). Implications of these findings to the taxonomy and genetic management of species in genus Warburgia are discussed.
Development of a genetic linkage map and quantitative trait loci (QTL) analysis in cowpea (vigna unguiculata (L.) walp)
(2010-06) Andargie, Mebeaselassie; Muluvi, Geoffrey M.; Pasquet, Remy S.
Cowpea is a diploid plant species which contributes significantly to food security in developing countries, especially in Africa. This research project was carried out in view of the upcoming introduction of Bt cowpea in Africa which is likely to alter the equilibrium existing within the cowpea taxa. The objectives of this study were to develop viable microsatellite markers and construct the SSR based linkage map, identify quantitative trait loci that regulate yield, domestication related traits as well as flower scent and identify the volatile compounds that attract pollinators to cowpea flowers. In order to achieve these goals 159 F7 recombinant inbred lines including the two parents and 206 markers (202 SSRs and 4 morphological) were used. The first SSR based linkage map of cowpea was constructed that spans a genetic distance of2991cM. QTL for seed weight (SW), domestication related traits (DRT), flower scent/aroma were mapped in all 159 F7 plants and the two parents 524B x 219-01. Six QTL associated with 74 % of the phenotypic variance were detected for SW on chromosomes 1, 2, 3 and 10. Both the 524B and 219-01 alleles increased SWat six of the QTL on chromosomes 1, 2, 3 and 10. For domestication related traits, nine QTL (four for testa size and five for pod fiber thickness layer) explaining 54.5 and 47.9 % of the phenotypic variance, respectively were on chromosomes 1,2, 4, 6, 7 and 10. The 524B allele increased DRTs at three-fourth of all QTL. QTL for SW and DRTs were clustered on chromosomes 1 and 10. Association of SW and DRTs QTL may be the cause of the significant phenotype and genotypic correlation detected between the two traits. The test of linkage vs pleiotropy for SW and DRT QTL on chromosomes 1 and 10 suggested pleiotropy. For flower scent/aroma, 63 QTL were detected on chromosomes 1,2,3,4, 5, 6, 7, 8, and 10. In addition, a total of twenty-two different volatiles were identified by the GS-MS technique. Clustering ofQTL were observed on chromosomes 1,2, and 4 mainly, suggesting that it can occur either due to the presence of a single locus with pleiotropic effects on several volatiles or as a result of tightly linked different loci. Such loci may encode transcription factors that co-ordinately regulate genes, or they may encode enzymes that catalyse limiting steps in single pathways. It is anticipated that this resource will have an important impact towards the development of marker assisted selection systems for the cowpea breeding community, and for future genetic studies in cowpea.
Morphological and genetic diversity of the new invasive species bactrocera invadens (diptera: Tephritidae) in Africa
(2011-08-02) Khamis, F. M.
Species of the genus Bactrocera in the family Tephritidae, or `true fruit flies', are among the most important pests of fruits and vegetables. Because of the broad larval host range and cosmopolitan distribution, pest fruit flies are highly invasive, with adults often exhibiting a strong tendency for dispersal and larvae being readily transported to new areas via fruit movement. Recently, a pest species was recorded for the first time in Kenya and has subsequently been found in countries across tropical Africa. The insect was described as Bactrocera invadens, due to its rapid invasion of the African continent. Taxonomic description of the B. invadens depicted different thoracic colourations that are morphotypes of the same pest. As invasive species spread around the globe, it is becoming increasingly evident that a detailed knowledge of the biology, genetic structure and geographical variability, of a given species is a prerequisite to planning strategies for quarantine, control or eradication. In this study, the morphometry, population structure and the genetic variability of different populations of the B. invadens distributed across the actual species range of tropical Africa and a sample from the presumed aboriginal home of Sri Lanka was investigated. Morphometry using wing veins and tibia lengths were used to separate the morphotypes of the B. invadens and a comparison to other closely related Bactrocera species. Fourteen distances between 15 selected landmarks on the wing were computed to characterize the shape and size of the wings for differentiation. Bactrocera invadens from all the localities analysed could not be separated by both the Principal component and canonical variate analyses. These two parameters separated the B. invadens from B. correcta, B. cucurbitae, B. oleae and B. zonata and it clustered together with B. dorsalis and B. kandiensis. Eleven polymorphic microsatellite loci were isolated and characterized and used for population genetic studies. The polymorphism of these loci was tested in individual flies from two natural populations (Sri Lanka and Democratic Republic of Congo). Allele number per locus ranged from three to 15 and eight loci displayed a polymorphic information content greater than 0.5. These markers were used to address three major issues on 13 geographic populations of the B. invadens, namely: (i) The historical origin of the B. invadens, (ii) the origin of the invasion of the B. invadens in Africa, and (iii) the extent of the establishment of the B. invadens populations in Africa and the genetic variability of these populations. Few years after its discovery in East Africa and its identification as a member of Bactrocera dorsalis complex, B. invadens samples from all equatorial Africa showed a high level of genetic diversity associated with an evident absence of geographic structure. These features are indicative of processes of rapid population growth and expansion with possible multiple introductions. The association of these two aspects reveals that B. invadens was not an indigenous African species that remained undetected for long period of time. Instead this species is a recent and an aggressive invader. Data from the timing of historical records, that indicated this insect as a new entrant in a region, are in this case concordant with the chronology of the spread as documented in this study. DNA barcoding using the COI gene was used to investigate the identity and species integrity of the B. invadens by comparing it with other Bactrocera species such as B. correcta, B. cucurbitae, B. dorsalis, B. kandiensis, B. oleae, B paraverbascifoliae and B. zonata.
Field studies influence of environmental factors and susceptibility to trypanosomosis of the Boran and Boran-N'dama crossbreed cattle in Kenya
(2011-08-04) Munga, Karongo Leonard
Trypanosomosis is a disease of humans and domestic animals caused by protozoan parasites of the genus Trypanosoma and mainly transmitted by tsetse flies (Glossina). The current methods for trypanosomosis control include vector control, treatment with trypanocidal drugs and keeping trypanotolerant livestock. The main advantage of keeping trypanotolerant animals is that it requires realtively lower recurrent expenditures, and is therefore suitable for low income production systems. The N'Dama of West Africa is the most important among trypanotolerant cattle, and some degree of trypanotolerance has also been observed in the Orma Boran of East Africa. The N'Dama is widely used in West Africa but there is no information on the potential value of the N'Dama in East Africa. In addition, there is lack of information on how the environmental conditions influence trypanosomosis in cattle. The objectives of this study were to compare infection rates, disease severity, treatment requirement and the ability of Kenya Boran and their crossbreeds with N'Dama cattle to survive under natural tsetse challenge and to determine hoe the preveiling environmental conditions influenced the disease in cattle. The study animals consisted of 335 cattle of four cattle breeds, namely Orma Boran (OB), Kenya Boran (KB), F1 N'Dama X Kenya Boran (NB) and backcross of Kenya Boran on F1 N'Dama X Kenya Boran. The cattle were produced in a tsetse free area and taken to the field site in Narok in six successive groups which were exposed to high tsetse challenge for at least one year. The animals were examined regularly for trypanosome infection, development of anaemia, growth rate and treatment requirement. The result of this study showed that increase ambient temperature was significantly associated with increase disease incidence in cattle while inreased normalised difference vegetation index (NDVI) had negative correlation with trypanosome incidence and persistence of parasitaemia in the cattle. Among the four cattle genotypes, the F1 N'Dama X Kenya Boran was the least susceptible. The orma Baran was less susceptible than the Kenya Boran and the backcross of the Kenya Boran. It was concluded that the N'Dama could play a role in enhancing trypanotolerance in the Kenya Boran cattle and the Orma Boran was more suitable that the Kenya Boran for crossbreeding with the N'Dama. It was also recommended that in development of livestock breeds for the tsetse infected arid and semi arid areas of Kenya should take into considerable the environmental conditions in these areas.
Agrobacterium tumefacients mediated introgression of Nicotiniana Protein Kinase (NPK1) gene in selected Kenyan maize genotypes to enhance drought tolerance
(2011-08-12) Muoma, John Vincent Omondi; Machuka, Jesse; Geoffrey Muluvi
In the last decade, the production of maize has gone down because of biotic and abiotic constraints, and this has resulted to prevalent famine. Of the abiotic stresses, drought is the most important stress affecting productivity of maize in Africa leading to up to 70% crop loss and in certain cases total crop loss. Conventional breeding, molecular marker assisted breeding and genetic engineering have already had, and will continue to have, important roles in maize improvement. The rapidly expanding information from genomics and genetics combined with improved genetic engineering technologies offer a wide range of possibilities for enhanced maize production. Genetic engineering of plants has been achieved through direct uptake of naked DNA into target cells and via Agrobacterium mediated transformation. Agrobacterium mediated transformation is increasingly becoming the method of choice due to its ability to generate transformed events containing low copy insertions. However this mode of maize transformation is dependant on genotype, age and physiological condition of the target explant and the infecting strain of A. tumefaciens. For every successful transformation protocol a reproducible regeneration system and transformation by a reporter gene is a necessity. The optimal regeneration condition for the shoot tips and immature zygotic embryos was observed to be 9µM 2, 4-D, 8.88µM BAP supplemented with 296µM adenine and 9µM 2, 4-D respectively for calli maintenance and shoot induction. Root induction in case of shoot apices was alleviated by the use of 1.97µM indole-3- butyric acid while immature zygotic embryos readily formed roots on MS without hormone after a maturation step. Transient expression of GUS was used to assay the explants for transformation frequency considering embryogenic calli formation, shoot induction and root formation had been optimized in the regeneration step. In these experiments, 10 days old seedlings shoot apex derived calli exhibited GUS activity at a transformation frequency (TF) of 0-4.2% while the 15 days old immature zygotic embryos derived calli exhibited a higher TF of 613% GUS activity making immature zygotic embryos better explant for transformation of the selected genotypes. Immature zygotic embryos were thus preferably transformed for drought with a gene that codes for an upstream transcription signal factor MAPKKK cascade (NPK1) triggered under drought stress (DS). The transformation efficiency for the four genotypes was TL08 0.79%, DHO1 4.87%, DLC1 2.64% and PTL001 5.35%. The seeds of the transgenic events were harvested, planted and both DNA and RNA extracted from the T1 events for southern, northern, and RT-PCR analysis to check on copy numbers and expression levels of the NPK1 gene. The T1 plantlets of tropical inbred TL08-(2)4, single hybrid cross of a PTL001, a multiple cross hybrid DHO1 and a dry land cultivar DLC 1 genotypes were planted in the green house and assessed for morphological and physiological changes associated with increase in DS tolerance when under water stress condition. The results showed that NPK1 effectively enhanced drought tolerance in TL08-(2)4 and PTL001, and there was no significant morphological difference between transgenic controls (well watered) and transgenic tests (subjected to moderate drought stress) using Turkeys Kramer HSD (p<0.05). Overall, there was between 20-35% enhancements of yield on comparison of the transgenic stressed events with non-transgenic stressed control
The causes and effects of pica habit of pregnant women in Msambweni division, Kwale District, coast province
(2011-08-12) Kinyua, Felix
Pica, the habit of eating non-food substances is a worldwide behaviour that is not limited to any geographic area, sex or status. It is common in retarded children and adults with some forms of insanity, hysteria and pregnancy. Geophagia is a form of pica where individuals consume soil and its products. Though the aetiology of Geophagia is not well elucidated, it could result more from habit, culture and superstition than from need for specific nutrients. Investigations to establish a cultural link to Geophagia among the Digo community revealed that 28% of women gave clay to their young children who wanted to taste what the mother ate. Thereafter, children ate clay as food. In clinical practice Geophagia is thought to occur due to iron deficiency anaemia. To establish the causes and effects of the habit in adults, 236 women were recruited for the study. 182 were pregnant geophageous and non geophageous test subjects. 54 were non-pregnant geophageous and non geophageous comparative study subjects. Through a questionnaire, commonly used substances and reasons for ingestion were investigated. The most common substance in the study area was found to be a synthetic product which is imported from Egypt, India and Singapore either as Portland cement or basmati rice and locally sold as roasted clay with herbs and spices. The substance is highly addictive and after long use, it kills appetite for other substances except itself. Investigations for anaemia showed that the substance is associated with high Hb, PCV and MCHC values that subsequently decline leading to chronic anaemia. However, anaemia even with active worm infestation was not able to cause Geophagia. Microbial analysis of the samples indicated contamination with air and faecal-borne bacteria including B.subtilis, P.mirabilis and Str. faecalis. Anti-microbial susceptibility tests showed various reactions to different drugs. Analysis of the trace element profile for local and imported soil substances indicated the presence of high levels of essential and radioactive elements which exposed photographic film. The latter elements include titanium, rubidium, strontium, zirconium, and niobium. The classic radio-nuclides that include uranium-238, thorium-232 and potassium-40 were also detected in significant quantities. All results were shown in text, tables and histograms where applicable
Use of a non mist propagation system to vegetavely propagate 12 varieties of E.grandis X E. camadulensis hybrids
(2011-08-12) Mwaniki, Fiona Nyawira
A series of nursery experiments was carried out to assess the effects of rooting medium (sand and clay subsoil, mixed in the following ratios 1:0, 1:1, 1:2, 0:1 and 2:1 respectively by volume), auxin concentration (0%, 0.6%, 0.8%, and 1 % IBA) and leaf area of cuttings (0, 30, 40, 50, 60, 80 and 100cm2) on rooting success of juvenile cuttings of Eucalyptus grandis x camadulensis hybrids. The Eucalyptus hybrids (EGC) cuttings were harvested from 5-year-old ramets, and propagated in non mist poly-tunnels, which act as propagation chambers. Among the treatments experimented, EGCs accounted for 8.6% and 14.4% of the total variability in rooting and shooting, respectively. The rest were accounted for by rooting media, IBA concentrations and their respective interactions. Results from logistic analysis carried out individually for all EGCs showed no significant interaction effect of IBA and media on the rooting of the 1 2 EGC hybrids tested. This indicates that EGC hybrids did not require the application of exogenous IBA for rooting. Cuttings without application of IBA rooted with highest rooting observed in clay sub soil (65.0%). The overall effect of propagation media was significant (p<0.01) on rooting and shooting percentage, with clay sub soil having the highest mean rooting (59.9%) and shooting percentages (81.9%) compared to sand soil which had the least mean rooting (12.5%) and shooting percentages (23.0%) among different EGCs. Leaf area had a pronounced effect on rooting and shooting percentage of EGC hybrids, with leaf area of 100cm2 giving the highest rooting (65.9%) and shooting (78.1%). Leaf area of Ocm2 gave the lowest rooting (7.5%) and shooting percentages (12.7%) and least number of roots and shoots in sand soil. On the basis of these results, EGC hybrids can be successfully propagated in a non mist poly tunnel. Clay sub soil, in combination with a leaf area of 100 cm 2 and exclusion of the use of IBA is recommended in this propagation system. Unlike mist propagation chambers, non mist poly tunnels are a cost effective method of propagating EGC hybrids and the exclusion of IBA as deduced from results of this study will further reduce production costs. In addition, this method uses materials that are readily available and can be used in rural areas which are without electricity and in limited water supply
Gene pool organization, population genetics and wild-crop complex dynamics of cowpea (vigna unguiculata)
(2011-08-12) Eric, Bertrand Kouam; Muluvi, Geoffrey M.; Pasquet, Remy S.
Cowpea is a tropical legume contributing significantly to food security in developing countries, especially in Africa. This research project was motivated by the upcoming introduction of Bt cowpea in Africa, likely to disturb the genetic equilibrium existing between wild and cultivated cowpea. The objectives of this study were to assess the genetic relationship between wild and cultivated cowpea, to determine how inbred and how differentiated are cowpea populations as well as gene flow, to estimate the mating system parameters in cowpea and their relation to ecological agents. In order to achieve these goals, wild and cultivated cowpea individuals were electrophoreticafy analysed using isoenzymes and PCR-RFLP techniques. Isozyme analysis revealed 61 alleles within wilds, 30 in cultivated and 29 common while chloroplast DNA variation was evident with two distinct haplotypes, the two within wilds, one in cultivated and one common. Very little genetic variation was found within accessions. Cultivated cowpea was found to be less diverse compared to its wild progenitor. Within wilds, genetic diversity indices decreased significantly from Southern to Western Africa via Eastern and Central Africa. Cultivated cowpeas were highly characterised by three markers (cpDNA haplotype l, Amp2l02 and Fle3096). Positive and significant correlations were found between any pair of these markers within wilds. Wild cowpas from West Africa region and especially Ghana at the country level displayed significantly most of these markers. Nei’s genetic distances indicated wild accessions from western Africa more close to the domesticated taxa. These results indicate a possible centre of domestication of cowpea in West Africa. Populations of cowpea showed high level of genetic differentiation, suggesting a low level of gene flow between populations. Gene flow estimation between populations based on the level of population differentiation and private allele's frequencies were confirmed to be low. The inbreeding coefficients were significantly high, highlighting a significant departure from Hardy-Weinberg expectations. Genetic investigation of the mating system in the region indicated up to 97% self-fertilization. Most of the genetic variation (65.8%) was found between populations. Isolation by distance either based on the level of population differentiation or genetic distance was positive and significant, (r = 0.142, P < 0.001) and (r = 0.2318, P < 0.001) respectively. Outcrossing rates were high in Kenya (East Africa) (32.7%) compared to West Africa (3.4%). The monthly outcrossing rate estimates over two years showed the same tendency in coastal Kenya, although a significant difference in mean was found between the two years. Significant level of crossing was found between relatives. The results indicate that wild cowpea have a mixed mating system primarily inbred. The monthly specific outcrossing rate was inversely correlated (r = - 0.68, P < 0.001) with rainfall distribution and positively correlated with flower density (r= 0.44, P = 0.069) and temperature (r = 0.50, P < 0.050). These data point out the dynamics of the mating system in cowpea through time. Implications of all these results in the light of releasing genetically modified cowpea, genetic conservation management and breeding programs in cowpea are discussed
Somatic embryogenesis and agrobacterium-mediated transformation of selected maize (zea mays.L) genotypes in Kenya
(2011-08-12) Ombori, O.; Machuka, Jesse; Gitonga, Nkanata Mburugu
Maize (Zea mays L.) is one of the most important staple human food crop for over 90% of Kenya's population. However production has lagged behind due to drought, pests, diseases, Striga and low soil fertility despite much effort put in using conventional breeding methods. An efficient in vitro regeneration system is an important step for genetic improvement of maize against production constraints. Biotechnology offers an impressive option to supplement the ongoing efforts on developing genetically enhanced germplasm for achieving sustainable food production. This study was done with the objectives of assessing the regenerative capacity and assessing the genetic transformation ability of tropical Kenyan maize genotypes. Somatic embryogenesis and plant regeneration was achieved from immature maize embryos. Callus was initiated on N6 medium supplemented with different concentrations of 2,4-D, 3% sucrose, 10 mgl-1 silver nitrate, 100 mgl-1 casein hydrolysate and 2.875 mgl-1 proline. The concentration of 2,4-D, genotype and age of embryos had a significant effect (p<0.5) on the percentage of primary and embryogenic callus formed. The induction of primary callus ranged between 0 and 97% and embryogenic callus from 0 to 84%. Somatic embryos were matured on N6 medium supplemented with 6% sucrose and 1 mgl-1 NAA. Regeneration was achieved using MS medium supplemented with 3% sucrose. Plants formed from maize genotypes ranged between 0 and 12 per culture vessel. Callus initiation and plant regeneration were genotype dependent. Maize lines H627 and CML216 had the highest mean number of shoots formed. Plantlets were transferred into half MS medium supplemented with IBA for enhancement of root development. In vitro regenerated plants were successfully transferred into pots in the greenhouse and into the field and they grew to maturity and set seeds in Ro and R1 generations. Transgenic plants expressing Bar and Gus genes employing the Agrobacterium-mediated transformation technique were obtained. Agrobacterium-mediated transformation technique was used for the introduction of a Gus reporter and bar into the immature embryos and embryogenic callus. Transformation experiments were carried using Agrobacterium strains EHA101 containing pTF102 binary vector, EH10I, AGL1 and LBA4404 containing pBECK2004, LBA4404, GV and EHA105 containing pCAMBIA2301 and AGL1 containing pSB223. The four vectors harboured a Gus gene. In this study transformation of the maize embryos was demonstrated, however the efficiency was low. Transient Gus activity was used as an initial step to assess if transformation had taken place. Transient Gus gene expression was confirmed by histochemical b-glucuronidase (Gus) activity on the 3`d day of co-cultivation of infected immature embryos and embryogenic callus. Transient Gus expression was also observed in the tissues of the putative transformants. Transient Gus gene expression was influenced by the co-cultivation period, genotype and Agrobacterium strain. Co-cultivation on the third day realized the highest proportion of embryos showing transient gene expression as compared to the fourth day of co-cultivation. Agrobacterium strains EHA105 containing pCAMBIA2301 and EHA101 containing pTF102 vector were more effective in producing high transient Gus gene expression. Despite EHA 105(pCAMBIA2301) producing the highest transient gene expression in immature embryos and embryogenic callus, plants were not regenerated. PCR amplification of bar and Gus gene confirmed the transfer of the transgenes. Southern blot hybridization confirmed the integration of the Gus gene into maize genome. In conclusion, the study established a reproducible regeneration and transformation system from immature embryos of tropical maize genotypes
Genetic differentiation, vector competence to dengue 2 viruses and host preferences of coastal and inland aedes aegypti populations from Kenya
(2011-08-23) Burugu, Marion Warigia; Eucharia, U. Kenya; sang, Rosemary; Kamau, Luna
The mosquito, Aedes aegypti is the main vector of dengue viruses responsible for millions of dengue fever and hundred of thousands of dengue hemorrhagic fever cases annually in tropical regions of the world. At the Kenyan coast, dengue incidences continue to be reported while inland, the disease is not documented. Since Aedes aegypti is widely distributed in Kenya, characterization of patterns of gene flow and genetic differentiation between Ae. Aegypti populations from these regions was necessary. Additionally, the vector competence of these populations and their bloodmeal host preferences, which influence disease epidemiology need to be established before implementation of any control strategy. A hierarchical population genetic study was conducted among 30 Aedes aegypti populations from coast and inland parts of Kenya. Single-strand conformation polymorphism analysis was used to examine genetic variation in a 389 - base pair region of the NADH dehydrogenase subunit 4 mitochondria) DNA gene (ND4). Vector competence to dengue 2 viruses was examined using immunofluorescence procedure and host-feeding preference determined using enzyme linked immunoabsorbent assay technique. Fourteen haplotypes were identified from a total of 1005 individual Ae. aegypti. Phylogenetic analysis of these haplotypes indicated two maternal lineages. Gene flow estimates demonstrated that coastal populations were relatively panmictic implying that dispersal in search for oviposition sites and human activities are factors that shaped the genetic structure in this area. Inland populations exhibited relatively restricted gene flow especially between seasons (Nm <1), reflecting possible bottleneck effects between seasons. There was no barrier to gene flow and human movement may be contributing significantly to the observed levels of gene flow. Aedes aegypti from coast region were more susceptible to dengue 2 viruses (19.05 - 77.7 % infection rates) than those of inland region (10.26 - 46.2 %), however, this difference in infection rates was not statistically significant (X2 = 1.3659, p>0.05). Restricted gene flow and low infection rates of the inland collections may explain why there has been no documented outbreak or incidence of dengue viruses circulating at the inland region. A total of 147 individual mosquitoes were positive for different antisera, 65.3 % of which had fed on human as a single host. Mixed feeding was recorded in 19.04 % of the individuals, (89.28 %) with both humans and baboon blood. Overall, human blood index was 84.82 %. This shows that Ae. aegypti has high preference for human blood more than other hosts even in the presence of other hosts indicating the anthropophilic nature of this species. Overall low genetic diversity in ND4 gene in coastal and inland Kenya Aedes aegypti populations was demonstrated suggesting that these populations could be more ancestral in nature and have not undergone a major evolution over the years.
Determination of CD8+-cell responses in a high risk HIV negative population
(2011-08-23) Muturi, Margaret Wangui
Naturally acquired cellular immunity in individuals who have been exposed to HIV-1 but have remained uninfected may hold clues for the design of an effective HIV vaccine. IFNy Elispot has emerged as one of the widely used assay to monitor HIV-specific immune responses. It is becoming the assay of choice for evaluation of HIV-vaccine-induced cell-mediated immune responses in many clinical trials. The objective of this study was to investigate the CTL responses of high risk HIV seronegative individuals to HIV A and RENTA vaccine peptides. The study further sought to investigate whether it was possible to recruit, sample, counsel and follow-up a cohort of high risk seronegative volunteers over a duration time in preparation for vaccine trials. To achieve these objectives, 30 volunteers filled a questionnaire, were counseled, tested for HIV status, recruited and enrolled in a 15 month study.The thirty exposed seronegative (ES) volunteers reported frequent unprotected sex with people of unknown HIV-1 status at enrollment. Every 3 months the volunteers were seen at the KAVI Kangemi clinic where blood samples were taken for the determination of the CTL responses, their HIV status was re-checked, filled questionnaire to assess the changes in their risky sexual behaviour. It was possible to recruit and follow-up the 30 volunteers for the entire duration of the study. All the thirty samples did not show HIV-1 specific T cell responses to both RENTA and HIV-A peptides using the ex vivo Elispot assay during the four time points (months 0, 3, 6 and 9). To investigate whether these results were truly negative, samples from 5 seronegative discordant couples were used. There were no HIV-1 specific CD8+ IFNy T cell responses in the HIV negative spouse. To investigate whether the ex vivo Elispot was unable to detect the responses, cultured Elispot assay was applied to the samples. They all tested positive with variations between peptide pools and individuals. The fact that cultured Elispot detected the responses from the 5 seronegative spouses of HIV infected partners and from 12 of the thirty means that the ex vivo Elispot assay was not sensitive enough to detect responses to the tested vaccine peptides. Cultured Elispot expands the memory CTL thus enhancing the detection of the responses. Using this method it was possible to demonstrate that HIV-1 specific CD8+ IFNy T cell responses exist in high risk exposed seronegative individuals. Pool 90 gave positive responses with all the samples. It would appear that combining the pools of peptides would elicit consistent CD8+ IFNy T cell responses and therefore make a better vaccine candidate. The results suggest that there is need to exercise very stringent criteria for enrolling high risk exposed seronegative participants to any study group meant to investigate immunological parameters related to HIV exposure.
Respones of mosquitoes to heavy metals
(2011-12-15) Odhiambo, Mireji Paul
Investigations were conducted to determine the influence of heavy metals on urban mosquito populations spatial distribution and composition and Anopheles gambiae sensu stricto fitness. Differential induction of proteins as well as expression of metal lothionein, alpha tubulin gene expressions in third instar An. gambiae s.s larvae in response to heavy metals selection was also investigated. Heavy metal concentrations and mosquito larvae species composition in larval habitats of urban Kisumu and Malindi, Kenya were determined by atomic absorption spectroscopy (AAS) and taxonomic keys respectively. A susceptible strain of An. gambiae s.s third intar larvae was separately placed under selection pressure with cadmium, copper and lead at LC30 and control through five generations. Egg hatchability, fecundity and survivorship of the larvae and adults were monitored in the sixth generation in the absence of heavy metal selecting agent. First, third and fifth generation selection survivors were screened for expression of the metallothionein and alpha tubulin genes by semi quantitative RT PCR and for differentially expression proteins by 2D gel electrophoresis. Manganese and iron were the most prevalent heavy metals in larval habitats in urban Kisumu and Malindi respectively. Concentrations of most were above WHO acceptable limits for drinking water. Copper and lead concentrations significantly influenced presence of Ae. Aegypti, while presence of An. gambiae was affected by lead concentrations. Resistance to cadmium, copper and lead selection increased 13, 11 and 78 folds respectively between first and fifth generation. There was significant reduction in egg viability, larvae survivorship, pupation, magnitude of adult emergence, fecundity, and delay in pupation in the heavy metals selected populations relative to control. The innate rate of increase and mean generation time were significantly lower in heavy metal selected populations than control. Instantaneous birth rate was also significantly lower in all but lead selected populations, than control. Population doubling time was significantly higher in the heavy metals selected than in the non-selected control populations. Expression of metallothionein was significantly higher in cadmium and copper selected than control populations in third and fifth generations respectively. Expression of alpha tubulin was significantly higher in cadmium selected than in control populations in fifth generation. Most differentially expressed protein spots were acidic and of low molecular weight among all metals and generations. Type of heavy metals and generation (selection pressure) were main indicators of differential expression magnitude. Mosquitos that traditionally exclusively proliferate in clean water- like An. gambiae seems capable of expanding their niche into polluted habitats and other hostile environments, but at a considerable biological costs. Semi quantitative RT PCR was sufficiently sensitive to detect heavy metals resistance/ adaptation in An. gambiae s.s larvae in a metal, gene and selection pressure specific manner. Both genes display both qualitative and quantitative potential application in monitoring both An. gambiae s.s adaptation status to heavy metals selection as well as biomonitoring heavy metals pollution through larval habitat. 2D gel electrophoresis also appears sufficiently sensitive to detect heavy-metals and generation specific responsive genes in An. gambiae s.s larvae and can also be used to biomonitor heavy metals environmental anopheles adaptation to pollution. This process would greatly be facilitated by characterization of the spots by mass spectrometry and molecular functional analysis of the spots against An. gambiae protein database.
Studies on the Midgut Lectin Gene of Glossina Austeni
(2011-12-19) Amin, D. N.
Trypanosomiasis continues to be a serious problem in sub-saharan Africa. The inadequacies of existing control strategies necessitate the development of alternative control measures. In most cases, the causative agents of African trypanosomiasis, Trypanosoma spp., require an obligatory passage through the tsetse, Glossina spp (Diptera; Glossinidae), vector. This developmental step to the parasite leads to vector-parasite interactions, especially within the midgut. Among the vector molecules highly implicated in the interplay are carbohydrate-binding proteins known as lectins. This thesis describes studies on the midgut proteolytic lectin of Glossina austeni. The purification of a proteolytic lectin from the midgut homegenate of G. austeni was achieved through a 2-step chromatographic procedure, involving anion exchange and affinity columns. The purified protein was shown to cause the agglutination of Trypanosoma brucei brucei (Kinetoplastida, Trypanosomatidae) and washed rabbit red cells in vitro. It also showed some trypsin activity when the chromogenic substrate, chromozym-TRY, was used. Polyclonal antibodies to this molecule were used to isolate a pulative gene encoding the protein from a G. austeni midgut cDNA library. The sequence of the isolated gene (GenBank Accession Number DQ060150) showed very high similarity to another gene previously obtained from G. fuscipes fuscipes midgut cDNA library. Analysis of the gene sequence, using basic bioinformatics tools, showed that the translated protein with 274 amino acids contains a signal peptide region and signature motifs for the serine protease trypsin family. The recombinant protein was expressed in E. coli BL 21 (DE2) cells and the purified product used to raise polyclonal antibodies. The recombinant Glossina proteolytic lectin was further expressed in Spodoptera frugiperda (Sf) 21 cell lines by the baculovirus expression system. This is the first Glossina protein to be expressed with this system. The baculovirus-expressed recombinant lectin was found in the medium of baculovirus-infected Sf-21 cell cultures indicating that the tsetse fly-derived signal peptide was recognized and cleaved by the Sf-21 cells. The recombinant protein was purified by immuno-affinity chromatography and shown by periodic Acid Schiff stain to be post-translationally modified by glycosylation. Moreover, the glycosylation pattern was not via the classical O-linked and N-linked sugar attachment motifs as these were absent from the nucleotide sequence. Both the baculovirus- and bacteria-expressed lectin proteins showed agglutination and enzymatic properties. The two recombinant forms of the expressed lectins showed no significant differences in terms of biological activity indicating that the sugar moiety may not be crucial for these functions. It is plausible that the physiological relevance of the sugar moiety is to act in synergy with the signal peptide for proper targeting of the secreted protein and also enhancing its solubility within the midgut region of the fly. The findings provide an important contribution in the characterisation of Glossina proteolytic lectin. The constructed recombinant baculovirus, designated AcMNP.gpl, constitutes a very useful molecular tool for further investigation of both the structure of the protein as well as the mechanisms of action of Glossina proteolytic lectin (Gpl) upon African trypanosomes. Preliminary data from the present study has led to the suggestion that the initial part of interaction between Glossina proteolytic lectin and D-glucosamine, which is binding, may rely more on structural complementarities between the two molecules, in a pattern similar to a lock and key mechanism.
Isolation and characterization of variable antigen types (VATS) of trypanosoma evansi stock in Kenya
(2012-01-06) Ngaira, J M
Trypanosoma evansi is a haemoflagellate of veterinary importance that can infect most mammals but is generally more severe in camels and horses. It causes surra, a disease of great economic importance in Africa, Asia and South America. The standard laboratory method for diagnosis of surraa is to demonstrate and identify trypanosomes in the blood of the infected animal. Because parasitological techniques have low sensitivity, alternative methods are used to complement parasite detection. Serological methods are based on variable antigens of the trypanosome. T. evansi, like all African trypanosomes, has a surface coat composed of the variant surface glycoprotein (VSG) which acts as a defence against general innate immunity and against acquired immunity directed at invariant surface antigens. To counter specific antibodies against the VSG, trypanosomes undergo antigenic variation, the change to expression of a different VSG during which, distinct trypanosome populations are produced termed variable antigen types (VATs). Predominant VATs (pVATs) tend to appear regularly during the early stages of infection and can be used as diadnostic antigens. The T. evansi pVAT designated RoTat 1.2 was originally isolated from a buffalo in Indonesia and has been shown to be expressed in all T. evansi examined to date. CATT/T. evansi, an antibody detection test based on RoTat 1.2 VSG, has shown high specificity in various geographical regions of the world; false negative results have been observed but not investigated. No previous use of CATT/T. evansi has been reported in Kenya. Consequently, this study aimed to investigate the suitability of RoTat 1.2 VAT as a diagnostic antigen for T. evansi in camels in Kenya. In the survey conducted, 2227 camels were screened of which 2038 belonged to nomadic pastoralists in T. evansi endemic Isiolo District of Eastern Province. For comparison, parasite detection was used as the reference method and Suratex for the detection of circulating antigen. Results showed that CATT/T. evansi detected 35 of 51 parasitologically positive camels (68.6% sensitivity), and Suratex® 58.8% (30/51). Both tests were highly specific (100%). The overall prevalence was 2.3% (51/2227) by parasite detection, 32.2% (327/1017) by CATT/T. evansi and 19.6% (188/961) by Suratex®. It was concluded that CATT/T. evansi and Suratex® were able to detect aparasitaemic infections rapidly and were more sensitive than parasitological methods in revealing the true extent of trypanosomosis in a herd. A follow-up was conducted to investigate the false negative results of Three possible explainations were considered for the false negative results by CATT/T. evansi: firstly, in early infections, antibody levels are too low to be detected. Secondly, the RoTat 1.2 VSG gene may be absent in some T. evansi isolates. Thirdly, the RoTat 1.2 VSG gene may be present but not expressed. Serological examination included immune lysis, direct agglutination, immunoflouorescent antibody test (IFAT) and Western blot. The presence of the RoTat 1.2 VSG gene in the genome of the T.evansi trypanosomes was determined using two PCR tests, one targeting a 488-bp fragment and the other a 205 bp fragment of the RoTat 1.2 VSG gene. Expression of the RoTat 1.2 VSG was determined by Western blot analysis using specific anti RoTat 1.2 VAT antibody made in mouse. Results showed that of the 16 T. evansi isolates not detected by CATT/T. evansi, four lacked the RoTat 1.2 VSG gene and did not express isoVATs of RoTat 1.2. This group. designated Group 1, comprised JN 2118Hu, JN 6512Muh and JN 6524 Muh, JN 4306Moh and the reference T. evansi KETRI 2479. Group 2 T. evansi contained the RoTat 1.2 VSG gene not expressed. In Group 3, the RoTat 1.2 VSG gene was present and expressed, but no explaination was immediately found for CATT/T. evansi failing to detect this group of T. evansi. To characterize T. evansi that are undetected by RoTat 1.2, the VSG cDNA from T. evansi JN 2118Hu was cloned and the nucleotide sequence determined. A region of the sequence lacking similarity with already known trypanosome sequences was identified by GenBank homology search. A PCR test, targeting a 273-bp segment of JN 2118Hu gene, was developed which specifically identified all five non-RoTat 1.2 T. evansi tested. No amplification was observed in any of the 27 RoTat 1.2 positive T. evansi tested. All 10 T. brucei tested negative. In conclusion, this study showed that the diagnostic antigen RoTat 1.2 is specific for T. evansi, but lacks the potential to detect all T. evansi. Clearly, the RoTat 1.2 VSG gene may be sufficient, but not necessary to identify a parasite at T. evansi. This study has provided valuable information for the future development of a practical serological test for the sensitive and specific detection of T. evansi infections.
Studies on indigenous bacillus thuringiensis isolates active against selected insect pests
(2012-02-06) Okech, Matilda A.; Mwatha, W. E.
The overall goal was to contribute to crop-productivity by developing sustainable pest management and the objective was to select and identify and efficacious local B. thuringiensis isolate active against the selected stem borers, C. partellus, E. saccharina, B. fusca and S. calamistis. Various isolates of B. thuringiensis were isolated from soils and dead insects collected from different places in Kenya. They were screened against various stem borers including C. partellus, E. saccharina, S. calamistis and B. fusca. Based on the relative potencies, seven isolates (ICIPE 001, 012, 023, 027, 040, 054 and 061 were screened further and their LT50 value determined. Three isolates, ICIPE 012, ICIPE 023 and ICIPE 054 were selected from the seven isolates shown above. These three, had LT50 values ranging from 1.40 to 1.59 days, tested against C. partellus and were chosen as being most active. The LC50 values of these three isolates were from 0.32 to 1.6 x 107 spores/ml on C. partellus, from 0.09 to 0.15 x 107 spores/ml on E. saccharina and from 0.4 to 1.8 x 107 spores/ml on S. calamistis. By the value LC50 isolate ICIPE 023 was then selected for further studies (0.32 x 107 spores/ml). These three isolates were serologically identified as being, B. thuringiensis var. kurstaki. To establish differences among these three isolates, biochemical test using API analytical profile was carried out. The results showed differences in their reaction to citrate utilization, urea production and production or arginine dihydrolase. Only one of the isolates, ICIPE 023, was found to be different from the others in terms of composition of the cell wall proteins. From the LC50 values of the three isolates, ICIPE 023 was found to be more active against C. partellus larvae than ICIPE 012 and ICIPE 054. This isolate was selected for all the subsequent experiments. Using local raw materials, eight different media were formulated and tested for their support for growth, sporulation and -endotoxin production of the selected B. thuringiensis isolate, ICIPE 023. This isolate grew best in one medium composed of cow-dung (3%) and soya (3 against %) giving LC50 value of 0.042 x 108 spores/ml, when the first instar of C. partellus larvae were tested. Optimization of growth conditions for this isolate yielded the most potent toxin when cultured in 50ml volume (in 250 ml Erlenmeyer flasks) and an agitation speed of 300 rpm. Several UV-protectants namely, Congo red, clay soil and molasses were mixed with B. thuringiensis isolate ICIPE 023 grown in the cow-dung/soya medium separately and exposed to sunlight. These were tested at intervals against C. partellus larvae. Results showed that isolate ICIPE 023 grown in the cow-dung/soya medium exposed to the sunlight without UV-protectant retained activity of approximately 60% mortality after 120 hours exposure to natural sunlight. Field trials were conducted to determine the effectiveness of isolate ICIPE 023 culture against C. partellus. Results showed no damage to the maize plants sprayed with isolate ICIPE 023 culture- broth but the infested non-B. thuringiensis -treated maize plants showed plant damage as evidenced by stem tunneling of approximately 29%. B. thuringiensis ICIPE 023 culture grown in the medium of cow dung/soya had a shelf-life of approximately one year and gave mortality of approximately 27% using a concentration of 0.025 ml/ml of the insect diet after one year of storage at room temperature (25°C). Samples stored in the cold room (4°C) gave 63.3% mortality against C. partellus after one year. This showed that there was very little loss of activity when kept in the cold room.
Use of RNA interference and overexpression of purple acid phosphatase genes in management of parasitic plants
(2012-03-23) Alakonya, Amos E.; Machuka, Jesse; Monda, E. O.
Parasitic plants are major contributors to food insecurity in poverty stricken Sub Saharan Africa; To date, there is no effective parasitic plant control strategy that has been adopted by the majority of small scale farmers in the region leading to continued parasite spread and hence food insecurity. This study evaluated a variety of strategies against the parasitic plants Cuscuta pentagona, Striga hermonthica and Orobanche eaegyptica. The efficacy of RNA interference and intercropping with phosphorus efficient species was evaluated against C. pentagona and S. hermonthica. Further the effect of overexpressing purple acid phosphatases genes in tomato on 0. eaegyptica management was also evaluated. First, this study established that C. pentagona KNOX genes were involved in haustoria development. Targeting C. pentagona KNOX genes by interspecific RNA silencing through transgenic Nicotiana tabaccum as the host caused haustoria distortion and suppressed C. pentagona growth. Contrary to the results obtained in C. pentagona KNOX gene silencing study, targeting of the C. pentagona plasma membrane H+-ATPase through transgenic Medicago sativa did not suppress C. pentagona nor silence the H+ATPase gene. Instead, the parasite elicited a hypersensitive reaction and changed its mode of obtaining nutrients from symplastic to apoplastic transfer. In addition, this study showed that intercropping of maize with a P fixing legume Lupinus albus does not mobilize enough phosphorus in the rhizosphere for access by maize although it enhances cereal biomass accumulation and suppression of S. hermonthica emergence. Furthermore, it was shown that intercropping L. alb us and maize does not affect arbuscular mychorrhiza fungi colonization in maize roots. Of further interest was the confirmation that soil phosphorus level and arbuscular mychorrhiza fungi colonization have an inverse relationship in S. hermonthica tolerant and susceptible maize and sorghum cultivars evaluated. Finally, the overexpression of purple acid phosphatases from L. albus and Medicago truncatula in tomato resulted in low Orobanche emergence, enhanced root branching, improved tomato vigor and low arbuscular mychorrhiza fungi colonization. It was concluded that RNA interference, intercropping of maize with phosphorus efficient species like L. albus and overexpression of purple acid phosphatases from L. albus and M truncatula can reduce parasitic plant infestation and establishment. These findings lay the foundation for further studies in food crops like maize and Sorghum which have been greatly impacted by Striga.
Distribution importance and management of stemborers (lepidoptera) in maize production systems of semi-arid Eastern Kenya with emphasis on biological control
(2012-04-05) Songa, J. M.
The bulk of maize, which is the main staple food in semi-arid Eastern Kenya, is grown by subsistence small-scale farmers, and the yields are usually low, averaging 500kg/ha Stemborers are one of the major causes of these low yields. The general goal of this project was to generate basic information for the development of a sustainable biological control programme for stemborers in this region, with emphasis on parasitoids. In a survey conducted in six agroecological zones (AEZs) in March-June, 1996, the pests reported to damage maize were: chafer grubs, stemborers, termites, 'red ants', yellow necked spur fowls, ground squirrels, monkeys, porcupines, rats wild pigs and storage insect pests. Squirrels were considered to be the most widely distributed and important vertebrate pest of maize in all the study zones. Stemborers were reported as pests of maize in all the zones, and ranked first among insect pests in AEZs: UM4, LM4, LM3, LM4 and second in LM5, but were considered unimportant in UM2. Agronomic practices that may influence stemborer infestation in maize, including cropping systems, varieties, sowing time, fertilizer/manure use, stover storage, usage and disposal are discussed. Farmers use insecticides, wood ash, soil, saw dust, chilli pepper, dry cell powder and Tagetes minuta to control stemborers. The spatial and temporal distribution of stemborers, and the incidence and severity of stemborer infestation in maize, was investigated in farmers' fields in six AEZs during the LR 1996 and SR 1996/97. The stemborers that infested maize in the six AEZs were: Chilo partellus, Sesamia calamistis, Cryptophlebia leucotreta and Busseola fusca. C. partellus was the most abundant and widely distributed, was most prevalent in the lower altitude zones, whereas S. calamistis and C. leucotreta were more common in the higher altitude zones. C. partellus was mainly responsible for the early to late season infestation of maize in the LM3-5 zones, while C. leucotreta and S. calamistis infested the crop mostly from mid-season especially in the UM2-4 zones. Stemborer infestion was much lower than expected for this region (1-22%), and was attributed tothe unusually low rainfall received during the two seasons. Stemborers were found to be a potential major problem to maize production in all the zones, except in UM2, which had the lowest severity of infestation (1.64). A study was conducted to determine the interrelationships among stemborer density, damage and plant growth variables, at the vegetative, reproductive and maturity stages and, their effect on grain yield in maize at Katumani, in the SR 1997/98 and LR 1998. Correlations and path coefficient analysis were used. The path analysis model, which accounted for 86% of the total variation in maize grain yield, showed that, the effects of larvae and damage variables, on the plant growth variables and grain yield, were primarily through damage caused at the vegetative stage. Among the plant growth variables, plant height at maturity, had the highest direct positive effect on grain yield. Among the damage variables, stem tunneling at the vegetative stage, had the higest indirect negative effect on grain yield, larvae at the vegetative stage caused a grain yield loss of about 3g per plant. Studies conducted at Katumani, Ithookwe and Kiboko, for four seasons (SR, 1996 - Lr 1998), revealed that, the stemborers that infested maize were C. partellus, S. calamistis, C. leucotreta and B. fusca, with C. partellus being the most abundant (71.3% - 92.89%) and widespread species, while B. fusca was the least common (0 - 0.12%). A complex of upto 22 different parasitoid species were recovered from the stemborers, and the parasitism was highest at Kiboko followed by Ithookwe and Katumani in descending order. The most common indigenous larval parasitoids were C. sesamiae and C. curvimaculatus, and the pupal ones were Pediobius furvus and Dentichasmias busseolae but their effect was low. During the season of introduction of the C. flavipes, (SR 1997) and the following (non-release) season, C. flavipes was responsible for the highest parasitism of stemborers in all zones (10.33% - 25.81%). This study showed that C. flavipes was able to locate, parasitize and successfully colonize stemborers (C. partellus, S. calamistis and C. leucotreta), at the three sites. Kiboko appeared to be the most suitable site for the survival of C. flavipes. Partial life table analysis of C. partellus showed that the highest mortality occurred on the 3rd and 4th instar larvae, and was mainly due to disappearance. However, key factor analysis showed that parasitism by C. flavipes on the 5th and 6th larval instars was the key factor that determined changes in the densities of C. partellus, during the period of this study. An on-farm study in the six AEZs from July, 1997 to April, 1998, showed that, Pennisetum purpureum grass was the most widespread and abundant wild host of C. partellus and S. calamistis throughout the study period, followed by Panicum maximum. C. partellus was the predominant species, with the highest number being recovered from S. versicolor (94.6%). Only larval parasitoids were recovered from C. partellus in S. versicolor, with C. flavipes causing the highest parasitism (13.64%).
Bovine interleukin-3: cloning production and gene expression in cattle infected with trypanosoma angolense
(2012-05-04) Mwangi, Simon Musyoka
Interleukin-3 (IL-3) is one of the cytokines that act during the early and late stages of blood cell formation. To enable the study of the role of IL-3 in bovine haemopoietic stem cell differentiation, the polymerase chain reaction (PCR) was used to amplify an IL-3 cDNA from first strand cDNAS's prepared peripheral blood mononuclear cells (PBMNC) from N'Dama and Boran cattle. An analysis of the cDNA sequence reveals that it contains a 432-nucleotide (nt) open reading frame which codes for 144 amino acids (aa) . Cleavage of the putative signal peptide consisting of the first 17 aa yields the mature form of the protein (14.5 kDa). Comparisons of the bovine IL-3 sequence shares 90.7, 55.8, and 51.9 percent nt identity, respectively, in the coding region, and 85.4, 35 and 27.7 percent aa identity, respectively. The availability in recombinant form of large quantities of the mature protein encoded by this cDNA has enabled studies on the role of the cytokine in bovine haemopoiesis. These studies have shown that bovine IL-3 is a multipotential colony stimulating factor (CSF) whose activities include stimulation of the in vitro formation from bone marrow cells of eosinopil, neutrophil and macrophage colonies and in the presence of erythropoietin, of burst forming unit-erythroid (BFU-E) and mixed colonies. The expression of IL-3 mRNA was evaluated by reverse transcriptase (RT PCR) in cells from several organs from healthy Boran and N'Dama cattle to determine the sites of IL-3 gene expression and to gain an understanding of how the expression of the gene is controlled in cattle. IL-3 mRNA could not be detected in unstimulated PBMNC, unsorted bone marrow mononuclear cells (BMMNC), BMMNC enriched for T lymphocytes, lymph node mononuclear cells (LNMNC) and pleen mononuclear cells (SPMNC). However, follwoing stimulation with ConA, large amounts of the transcript were detected in all the cells with the exception of unsorted BMMNC. Studies of the kinetic patterns of IL-3 mRNA accumulation in LNMNC and SPMNC revealed that the mRNA accumulates rapidly following ConA stimulation, peaks after 3-6 h of stimulation and thereafter declines. To determine if there are any shared regulatory elements between the bovine and human IL-3 genes that might explain the similar patterns of gene expression observed in this study, a DNA containing a portion of the 5' flanking region of the bovine IL-3 gene was amplified by PCR from a bovine genomic DNA library. A search for homology with known IL-3 sequences in the GenBank database reveals that the first 487 nt of this DNA share 93.3, 66.5, 65.6 and 57.4 perent identity, respectively, with the sequences of the 5' flanking regions of the ovine, human, Rhesus monkey and murine IL-3 genes. A computer-assisted analysis of the sequence reveals several potential regulatory motifs, which closely match regulatory elements of the promoter of the human IL-3 gene both in their nt composition and spatial distribution. The further characterization of these DNA motifs will contribute to the understanding of how the expression of the IL-3 gene is regulated cattle. The expression of IL-3 mRNA in LNMNC, PBMNC and SPMNC from Trypanosoma congolense-infected cattle was also investigated to determine if depressed IL-3 gene expression plays a role in the ineffective haemopoiesis observed during infections of cattle with the African trypanosomes. In both Boran and N'Dama cattle infected with Trypanosoma congolense, IL-3 mRNA expression was never detected in unstimulated cells at all times of assay. Following stimulation of the cells with ConA, the amounts of IL-3 mRNA induced in LNMNC and PBMNC from the infected cattle were equal to those induced in cells from the non-infected controls. However, the amounts induced in SPMNC from infected cattle were significantly lower than those induced in cells from control animals. In summary, results of these studies demonstrate that bovine IL-3 has similar biological activities as IL-3 from other species, and suggest that bovine IL-3 is an important growth factor in induced haemopoiesis in cattle. They also show that, although the capacity of bovine T lymphocytes to express the IL-3 gene might not be impaired at the cellular level during infections of cattle with T. congolense, overall the production of IL-3 might be affected due to reductions in IL-3 gene expression in the spleen initially, and possible in other lymphoid organs in long-term trypanosome infections.
Ecological and behavioural studies of mosquitoes in Mwea Tebere irrigation scheme Kirinyaga district Kenya with special reference to anopheles arabiensis (diptera; culicidae)
(2012-06-05) Rapuoda, Beth Awuor
During this investigation which took place between April 1989 and February 1991, an attempt was made to determine the impact of rice irrigation practices on 1 mosquito species diversity and their relative population density; 2 malaria infection rates in both the human and mosquito populations, The studies also investigated the relative importance and attractiveness if various hosts for different mosquito species. Mosquitoes were sampled from two study villages, Mbui Njeru and Mathangauta, which are located in the Mwea irrigation scheme. The two villages are also accessible throughout the year. Mosquito sampling was carried out in each of the four houses in each village. Sampling was replicated daily for seven days each month in every village. Sampling methods for adult mosquitoes included collections from daytime indoor and outdoor resting sites using battery powered aspirators and pyrethrum knock down spray. Mosquitoes entering houses for feeding or resting purposes for feeding or resting purposes were also collected using miniature light traps. Larvae were collected from flooded rice paddies and pools of stagnant water using standardized dipping methods. Mosquitoes collected by the various methods were identified to various species and recorded accordingly. The physiological status of females of the two commonly known malaria vector species namely, anopheles arabiensis and anopheles funestus, was determined on the basis of abdominal appearance. Salivary glands and midguts of individual mosquitoes from the two species were also dissected out and examined for malaria parasites in the sporozoite and oocyst stages, respectively. Gut contents of the fully fed females were also individually collected on filter paper for blood meal analysis. Parallel to observations on mosquito infections, samples of finger prick blood smears from the human population were also made to determine the malaria parasite prevalence. A total of ten mosquito species, which included An. arabiensis and An. funestus, the two known vectors of malaria were recorded from the study area. An. pharoensis which is suspeced to have some potential role for malaria transmission was among the species collected. An. rufipes was the predominant species among those collected from outdoor resting sites. Monthly numbers of mosquitoes were significantly different with a definite peak recorded during the month of September. From the results obtained in this study it was evident that the fluctuation patterns of the vector mosquito numbers was greatly influenced by the rice growing cycle. The onset of preparation for rice nurseries particularly encouraged larval breeding. During harvesting period when water was drained from the paddies the relative numbers of An. arabiensis decreased. In Mathangauta village the two vector species alternated in their predominance. When the numbers of An. arabiensis was high that of An. funestus declined and vice versa. The increase in An. arabiensis coincided with the preparation of nurseries and seedling transplantation while the increase in An. funestus coincieded with the draining of water from the paddies and harvesting. Irrigation water released from paddies during harvesting found its way into drainage canals with dense submerged vegetation, thus forming suitable breeding sites for An. funestus. Variation in the numbers of An. arabiensis in Mbui Njeru was influenced by seasonal rainfall pattern in addition to rice cultivation cycle. Mosquito numbers were low during the rainy season, probably due to wash off effects of breeding sites by the rain water, but increased during the dry season. The mean counts between the two vector between the two vector species An. arabiensis and An. funestus were also significantly different with the former being the most abundant and present throughout the year. The species from both indoor and outdoor sites were also significantly different with more mosquitoes recorded indoors than outdoors. Surprisingly, it was also noted that the incidence of malaria was high when the relative numbers of An. arabiensis was low compared to the other months. It is therefore likely that the main vector responsible for transmission of malaria in Mwea Tebere is An. funestus. Investigations on the sporozoite rates of dissected female mosquito vectors showed that the monthly average sporozoite rate was not significantly different between the two villages (X2=0.303; P>0.05). The difference in the sporozoite rate with respect to the seasons was also not significant (X2=2.25; P>0.05). All the mosquitoes (n=4594) tested for sporozoite rate using the Enzyme-linked Immunosorbent Assay (ELISA) were negative. This confirmed earlier observations of low sporozoite rates through dissections of salivary glands. Studies on resting behaviour of female Anopheles arabiensis and Anopheles funestus showed that freshly fed mosquitoes preferred to rest indoors. A comparison of the resting behaviour of An. arabiensis and An. rufipes showed that fed females of the latter rested predominantly outdoors. Results on feeding behaviour determined through blood meal analysis showed that An. arabiensis fed predominantly on bovine hosts (79%). The difference in the numbers feeding on human and bovine hosts was significant (P<0.05) with more An. arabiensis feeding on bovine hosts. Malaria parasite prevalence rate in the human population for Mbui Njeru and Mathangauta was less than 8.7% for all age and sex groups. Plasmodium falciparum was the predominant species comprising 100% of the infections recorded. The malaria prevalence rate for Mbui Njeru was higher than that of Mathangauta village (F1,1=12.63; P<0.01) and all the other villages screened during the second year of this study. The peak malaria prevalence rate occurred in the month of July for both Mbui Njeru and Mathangauta villages. It was noted that the infection rate was higher in males (6.3%) than in females (3.7%) (F1,1=6.27; P<0.01). There was also significant difference in the malaria infection rate among the various age groups. The age group with the highest infection rate was the 10-14 year olds. The overall outcome of this study was that the numbers of mosquitoes were enough to maintain malaria transmission throughout the year in the area studied. However, the parasite infection rate in both vector and human population was lower than would be expected in the presence of such high numbers of vector mosquitoes. This situation was possibly due to the fact that the vector species were predominantly feeding on bovine hosts than on human beings is tendency by mosquitoes to feed more on animals than human beings is referred to as zoophily. Such feeding behaviour may form the basis of zooprophylaxis, an important method through which man-vector contact could be minimized. Zooprophylaxis is a practical malaria control method in which the community can participate by being encouraged to keep at least a few cows outside their houses. However, there is need for further studies to determine the optimal densities, directions and distances at which such barrier animals could be deployed, to ensure that they on the other hand do not worsen the situation by attracting large numbers of potential vectors to human habitations.
Studies on some reproductive physiological aspects of the Kenyan fruit bat, eldolon helvum (KERR, 1792) (Megachiroptera)
(2012-06-05) Simbauni, Jemimah A.
Cytochemical procedures have been used to study the anterior pituitary gland (adenohypophysis) of the Kenyan fruit bat Eidolon helvum (Kerr, 1792) during various reproductive phases, which comprise preovulatory, ovulatory, delayed implantation and pregnancy phases. The main purpose for these histophysiological investigations is to identify adenohypophyseal celltypes whose hormonal secretions directly or indirectlyinduce delayed implantation in the fruit bat under study. In a light-microscope study based on the size, shape and tinctorial affinities, six cell types have been identified in the adenohypophysis. There are three types of basophils notable for the presence of acid muccopolysaccharides in their cytoplasm, two types of acidophils notable for the presence of phospholipids and lipoproteins in their cytoplasm and the chromophobes which do not contain any stainable garnules. Periodic Acid Schiff's (PAS) reaction for carbohydrates has been used to distinguish between he basophils nad acidophils. All the PAS positive cells are referred to as basophils. PAS in combination with other dyes for example Alcian Blue have enabled further differentiation of basophils into three types, namely, type I basophil, type II basophil and type III basophil. Two types of acidophil cells in which carbohydrates are absent w ere identified namely: type I and II by their negative reaction with Period Acid Schiff's technique for mucoid substances and positive reaction with either Orange G or azocarmine B ('Azan Stain). The precise differentiation of the two acidophils is achieved by employing Brooke's (1964) method for lipids. The sixth celltype which lacks stainable granules was identified as the Chromophobe. The basophils type I, II and III were identified as Thyroid Stimulating Hormone (TSH), Follicle Stimulating Hormone (FSH), and Leutinizing Hormone (LH) cells respectivelyo n the basis of their tinctorial affinities. The adicophil cell type I and II were identified as somatotrophs (STH cells) and prolactin cells (PRL) respectively. Further the hormonal assays of progesterone, LH and prolactin at various reproductive phases was carried out by Radio-Immuno Assays (RIA) and Enzyme - Linked Immuno Assays (ELISA). The level of progesterone hormone was highest during pregnancy 56.45 ng/ml and lowest at delayed implantation period 1.626 ng/ml. The level of prolactin hormone was highest (7.67 ng/ml) during the period of delayed implantation and lowest 0.89 ng/ml at pre-ovulatory period. The level of LH was lowest during delayed implantation period (2.08 ng/ml). Electron microscopic (EM) studies on these cell types identified various cell organelles including nuclei, endoplasmic reticulum, mitochondria, ncleoli, Golgi apparatus, secretory vesicles etc. Six morphologically distinguishable cell types in the anterior pituitary gland of the female bat were identified. These cells were designated as type I-VI (TSH, FSH, LH, STH, PRL/LTH and chromophobes) on the basis of their secretory granules and other cytoplasmic organelles. When the female bats enter delayed implantation stage there is a remarkable rise in the number of granular prolactin cells (Brooke's method) with a concomitant depletion in the FSH and LH cells. These alterations in the adenohypophyseal cells are acoompanied by weight changes in the ovary and uterus. These observations suggest the involvement of prolactin in inducing delayed implantation. Hyperprolactinaemia suppresses gonadal function, probably by a short-loop inhibition of LH/FSH release. The mean values of the numbers of various types of cells observed and hormonal assays of LH, progesterone and prolaction during the different reproductive stages were calculated and the relationship between them determined using the chi-squared in a large table and regression techniques (Parker, 1979; Gomez and Gomez, 1984). Consequently, for the mean (In) cell and the mean (In) hormonal values, the standard errors were calculated. In E. helvum implantation occurs roughly 90 days after conception. During delayed implantation period the conceptus develops only as far as the bilaminar blastocyst stage. During this period, conspicuous changes occur in the endometrium, lining epithelium, and glands of the uterus. The reason for such a long delayed implantation period (±90 days from the zygote until implantation during the bilaminar blastocyst stage) can only be to ensure that the juveniles will leave the maternity at a time which coincides with the higher reainfall peaks when availability of fruits is at a maximum (Mutere, 1967). Moreover, since implantation is considered a promising target for contraception, the present study might help in future to define targets of attack for the development of new contraceptive agents for human use and welfare, taking into consideration the role of prolactin in both male and female subjects.

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