MST-Department of Microbiology

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    Analysis of Microbial Load And Diversity in Crickets (Gryllus Bimaculatus and Scapsipedus Marginatus) Used as A Source of Protein for Food
    (Kenyatta University, 2019-04) Jedida Wanjiru, Gatheru
    Crickets are drawing interest as sustainable source of protein for food and feed worldwide. However, there is little information on microbial quality of edible crickets thus the need for a profound insight into their safety. The objective of the study was to determine the microbial load of two edible cricket species Scapsipedus marginatus and Gryllus bimaculatus and to evaluate the efficiency of different processing methods (boiling, sun-drying, freeze-drying, snap freezing and deep frying) in reducing microbial counts. The wild-caught crickets were obtained from Nguruman (Kajiado County) and Mbita (Homa Bay County) while the farmed crickets were reared at Animal Rearing and Containment Unit at the International Centre of Insect Physiology and Ecology (icipe). The cricket samples were screened for microbiota using culture-dependent method. Fifteen adult crickets were pooled together and homogenized in Phosphate-buffered Saline (PBS). The solution was used for mycological and bacterial isolation and analysis. Bacterial microbiota was isolated using Nutrient agar (NA) and MacConkey agar (MCA). Fungal microbiota was isolated using Sabouraud Dextrose Agar (SDA) and Potato Dextrose Agar (PDA). The isolates were characterized morphologically and through sequencing of bacterial 16S SSU rRNA genes and fungal internal transcribed spacer (ITS) rRNA gene. Most of the bacterial isolates (70 %) on NA had characteristics typical of members of Bacillus spp. while on MCA, most bacteria (90 %) had characteristics suggestive of members of E. coli. Majority (80 %) of the fungal isolates on PDA had characteristics similar to those of Aspergillus spp. while most isolates (80 %) on SDA were members of Trichoderma spp. Microbial counts of fresh cricket samples were generally high, with bacterial population ranging from 2.5 × 104 - 3.8 x 104 CFU /g fresh weight. The fungal populations ranged from 2.0 × 104 – 3.0 × 104 CFU /g fresh weight. Upon processing the microbial counts reduced considerably with bacterial counts ranging from 0.1 x 102 – 0.8 x 102 CFU /g dry weight (boiled) and 0.7 x 103 – 2.2 x 103 CFU /g dry weight (sun dried). The fungal counts ranged from 0.7 x 102 - 0.9 x 102 CFU /g dry weight (boiled) and 0.9 x 103- 1.7 x103 CFU /g dry weight (sun-dried). Freeze-dried and deep fried samples had no microbial counts. The diversity of bacteria and fungi species in wild-caught crickets was considerably high compared to the lab-reared crickets with most isolates belonging to species pathogenic to humans. Phylogenetic analysis revealed that most bacterial isolates from the wild-caught crickets related to members of Bacillus spp. (57 %), Staphylococcus spp. (43 %) and E. coli (14 %). Fungal isolates related to Aspergillus spp. (57 %) and Penicillium roseopurpureum (29 %). From the farmed crickets, most of the bacterial isolates related to E. coli (60 %), Enterobacter (40 %) and Lactococcus garvieae (20 %). The fungal isolates related to Trichoderma asperellum (75 %), Aspergillus spp. (25 %) and Tetrapisispora fleetii (12.5 %). From the cricket samples processed by boiling and sun drying, the microbial diversity was very low with only two bacterial isolates related to Rickettsiella grylli (42 %) and Wolbachia spp. (29 %), while the two fungal isolates related to members of Aspergillus spp. (67 %) and Trichoderma asperellum (33 %). Thirteen potentially novel bacterial and fungal isolates from wild and reared crickets had no close matches from gene bank and need further investigation. This study shows that crickets harbor diverse microbial communities some of which are potentially pathogenic. Deep-frying, freeze drying and snap freezing completely eliminated bacterial and fungal contaminants thus minimizing microbial risks in crickets meant for food.
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    Antibiotic Susceptibility Pattern of Bacterial Uropathogens Isolated From Patients in Nakuru Level 5 Hospital, Kenya
    (Kenyatta University, 2017) Gachuhi, George Tibi
    Urinary tract infections (UTI) are bacterial infections encountered in the hospital and community and is preventable. It is among bacterial infection encountered with increasing antibiotic resistance to uropathogens, although there is availability of antibiotics. Despite the wide spread of antibiotics, it remains the common bacterial infections. Antibiotic susceptibility testing therefore provides information that allows clinicians to select the most appropriate antimicrobial drugs. Over the years, the UTIs antimicrobial resistance patterns have been changing. The study was carried out to establish the prevalence of bacterial isolates and their drug susceptibility patterns among the study population. A descriptive cross-sectional study was conducted in outpatients and inpatients presenting with symptoms of UTI. Purposeful sampling was used to obtain 385 respondents. Mid-stream urine sample were obtained from respondents using sterile bottles and bacterial isolates identification was done using biochemical tests. Culture and sensitivity pattern of uropathogens were determined using disc diffusion method. A questionnaire was administered to consenting respondents and data associated with risk factors was collected and analyzed at ɑ=0.05. Out of 385 urine samples 112 (29 %) patients were confirmed positive for UTI. The prevalence of UTI was higher among females (62.1 %) compared to males (37.9 %). Escherichia coli 66 (55 %) was the most predominant followed by Klebsiella pneumoniae 12 (10 %), coagulase negative staphylococci 25 (20.9 %), Staphylococcus aureus 11 (9.2 %) and Proteus mirabilis 6 (5 %). Antimicrobial profiles of E. coli strains showed the following susceptibility pattern to nitrofuratoin (100 %), cefotaxime (86.3 %), ciprofloxacin (83.3 %), gentamicin (81.7 %), ampicillin (45.3 %) nalidixic acid (48.5 %) and cotrimoxazole (44.1 %). Further 85% of the isolates were observed to be multidrug resistant, limiting treatment of UTIs with routinely used antibiotics. Hence, there is need for constant monitoring of antibiotic resistance for better management of patients on antibiotic treatment. In addition, the collected data could be use in determination of trends in antimicrobial susceptibility patterns and therefore assisting in policy formulation on the currently used antibiotics for management of UTIs.
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    Antimicrobial Activities and Bioassay Guided Identification and Bioactive Constituents of Selected Kenyan Medical Plants
    (Kenyatta University, 2022-01) Wanjiku, Kagika W; Sumesh Chander Chhabra
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    Antimicrobial activity and qualitative phytochemical composition of crude extracts from medicinal plants against selected enteric bacterial pathogens and candida albicans
    (Kenyatta University, 2016-09) Opinde, Hibert Rachuonyo
    Plant extracts with medicinal value have been used to treat many diseases that can either be bacterial, fungal or parasitic among many others. Plants with medicinal value produce certain chemical elements known as phytochemicals that have antimicrobial activity. Enteric bacterial pathogens are disease-causing microorganisms that are usually located in the intestinal tracts of either animal or human beings. The pathogenic members are usually associated with infections that are characterized by; enteric fevers, abdominal pain, diarrhoea, and vomiting. Candida albicans is a yeast fungus that is mainly found in the mucosal cavity of the vagina and intestinal tract as a normal microbiota but it can cause systematic infections in immunocompromised individuals. This study was aimed at determining the antimicrobial activity, combined effects of the selected plant leaf extracts of Tagetes minuta, Aloe secundiflora, Vernonia lasiopus and Bulbine frutescens against selected clinical isolates of Escherichia coli, Salmonella typhi, Staphylococcus aureus, Shigella flexineri, Enterococcus faecalis and Candida albicans obtained from Kenyatta University Health Centre; using the Kirby-Bauer method. In addition, qualitative analysis of the phytochemicals present in the extracts was also determined. The plants materials were obtained from Kenyatta University arboretum and identified by Taxonomist; Prof L.E. Newton and voucher specimen deposited in the University herbarium. The collected data was then analyzed in SAS version 9.1 using ANOVA and further subjected to a post hoc test with P<0.05 being considered significant. When used singly and in combinations against the test microorganisms the average zones of inhibition were found to be significant at P<0.05. When the plant extracts were used in low concentrations against the test microorganisms; Vernonia lasiopus was more active against Shigella flexineri (MIC 3.3μg/ml, MBC 7.1μg/ml), Bulbine frutescens against Shigella flexineri (MIC 3.2μg/ml, MBC 6.2μg/ml), Aloe secundiflora against Shigella flexineri (MIC 3.7μg/ml, MBC 8.0μg/ml) and Tagetes minuta against Enterococcus faecalis (MIC 5.1μg/ml, MBC 6.3μg/ml)*. The combining of the extracts also showed an increased and decreased antimicrobial activity with the interactions being significant; P<0.05. The average zone of inhibition formed by Aloe secundiflora and Tagetes minuta plant leaf extracts combination (8.67±1.86mm) showed a decrease in antimicrobial activity as compared to when Tagetes minuta (15.17±2.71mm) and Aloe secundiflora (17.00±2.10mm) respectively when used against Candida albicans. The qualitative phytochemical analysis showed the presence of four phytochemicals; saponins, tannins, alkaloids, and flavonoids. The study provides insight into the antimicrobial activities of the plant extracts and their use in the treatment of bacterial or fungal infections. This information might be used in herbal medicine in making concoctions to maximize their effectiveness. There is a need to elucidate the actual compounds in the plant leaf extracts responsible for the antimicrobial activity so that can be used in drug development.
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    Antimicrobial activity of some medicinal plants used by the Samburu community of Northen Kenya against selected bacterial and fungal pathogens.
    (2014-11) Onyambu, Abuga Samwel
    Medicinal plants form an integral social and cultural component, and sometimes the only alternative available treatment for health problems. Infectious diseases concern the whole world because they cause an estimated 98% of deaths in both children and adults in developing countries. The aim of the study is to determine ethnobotanical uses and the bioactivity of some medicinal plants used by the Samburu community against selected bacterial and fungal pathogens. This study evaluated both antibacterial and antifungal activity of the selected twenty two medicinal plants used by the Samburu community. An ethnobotanical survey was conducted and collected information from herbalists on their use in the treatment of various infectious diseases. Plants identification was done with assistant of a taxonomist from Kenyatta University. Voucher specimens were prepared and deposited at the Department of Plant Sciences herbarium. Methanolic plant extracts were screened using Kirby-Bauer disk diffusion technique against eleven strains of bacteria and fungi obtained from Centre for Microbiology Research at Kenya Medical Research Institute, Nairobi Kenya. They were either American Type Culture Collection (ATCC) or clinical isolates; Bacillus subtilis (clinical isolate), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 28910), Escherichia coli (ATCC 25922), Vibrio cholerae (ATCC 27622), Shigella dysenteriae (ATCC 26988), Candida parapsilosis (ATCC 18310), Cryptococcus neoformans (ATCC 19310), Aspergillus jlavus (clinical isolate), Microsporum gypseum (clinical isolate), Trichophyton mentagrophyte (clinical isolate). Minimum inhibitory concentrations (MICs) were determined by measuring the diameter around the discs, while micro-dilution technique was used to determine the minimum bacteriocidal and fungicidal concentrations (MBCsIMFCs). Extracts of Acacia nilotica, Thylachium africanum and Loranthus acaciae produced high antibacterial and antifungal activity results of ~9-15 mm and 9.375 - 18.75 mg/ml as showed by the MICs and MBCslMFCs results. Screening for phytochemicals; tannins, saponins, flavonoids, cardiac glycosides, alkaloids, and terpenoids indicated presence at varying concentrations either high, moderate, low or absent. Significant difference of zones of inhibition means of all the strains was observed at P:S;0.05. Clear indications from the data obtained were that these plants extracts serve as an enormous source of untapped antimicrobial agents. This study recommended that the Samburu community should continuously be sensitized on sustainable use of medicinal plants and that they should give priority to domesticating the medicinal plants. Further studies on the biological properties, isolation and identification of active components in the plant parts used in this research in order to test specific antimicrobial activity was also recommended.
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    Antimicrobial Susceptibility Profile of Neisseria Gonorrhoeae Isolated from Urethral Discharges and Men Who Have Sex with Men in Nairobi
    (Kenyatta University, 2018-12) Lewa, Pole Said
    Neisseria gonorrhoea has progressively developed resistance to previously used drugs and recently to fluoroquinolones. Currently, there have been reports of emergency of N. gonorrhoea strains with reduced susceptibility and some exhibiting extensive multidrug resistant to the cephalosporin class of antibiotics that remains to be the remaining option for management of gonorrhoea. In Kenya, published reports regarding the drug susceptibility of N. gonorrhoea is scanty because of lack of surveillance programs and reliance of syndromic management established 20 years ago. Currently, management of gonococcal infections still utilizes a fluoroquinolone in combination with a macrolide; a treatment option that was revised more than three years ago. This study was undertaken in order to determine the antimicrobial susceptibility profile of Neisseria gonorrhoea cultured from symptomatic men and asymptomatic men who have sex with men (MSM) attending Casino Special Treatment Centre (STC) clinic in Nairobi. A total of 264 participants were consecutively sampled of which 73 were symptomatic men patients that presented with urethral discharges and 191 were asymptomatic MSM during the period between September 2015 and August 2016. Swabs were directly inoculated on modified thayer martin agar plates and transported to the University of Nairobi department of medical microbiology laboratory in candle jars for analysis. Colonies that were suggestive of gonococci were identified and N. gonorrhoeae was confirmed using standard procedures. The Minimum Inhibitory Concentrations (MIC) using Etest for penicillin, tetracycline, ciprofloxacin, spectinomycin, erythromycin, azithromycin, gentamicin, cefixime and ceftriaxone were determined and WHO reference strains used as controls. Data analysis was done using SPSS and descriptive statistics used to analyse both demographic and risk sexual behaviour of MSM. The Chi-square test was used to determine level of significance age category and risk sexual behaviour. There was a significant association between age category and the variables of number of partners and insertive anal sex one MSM was engaged for the last 3 months. The overall isolation rate was 23.8% (63/264) where 20.1% (53/264) were recovered from symptomatic men and 3.7% (10/264) from symptomatic MSM. All N. gonorrhoea strains recovered urethral discharges were highly susceptible to cefixime, ceftriaxone and spectinomycin; gentamicin demonstrated moderate susceptibility (94.3%) levels. High resistance levels were observed in penicillin (49.1%), tetracycline (96.2%), ciprofloxacin (49.1%) and azithromycin 16.9%). Of the total 3.7% N. gonorrhoea strains isolates from asymptomatic MSM, 4/9 (1.5%) were recovered from the pharynx, 5/9 (1.9%) urethral and 0/9 from the rectal. All the gonococci isolated from the pharynx were highly susceptible to cefixime, ceftriaxone, spectinomycin and gentamicin (100%). Urethral strains were highly sensitive (100%) to spectinomycin and gentamicin only. Susceptibility to both cefixime and ceftriaxone was at 80% and 60% respectively. One strain showed high level MIC to both ceftriazone and cefixme; while one showed cefixime MIC increased level (≥2.0μg/ml) and (≥0.160μg/ml) respectively both recovered from the urethra. Cefixime and ceftriaxone currently used in the management of gonorrhoea are still effective in our local gonorrhoea conditions. The traditional antibiotics previously used to manage gonorrhoea are not effective and there is an emerging resistance of N. gonorrhoea strains towards azithromycin. There is therefore a need to conduct continuous surveillance of gonococcal strains and screen high risk group as they harbour gonococcal strains that are resistant to antibiotics. The study recommends molecular characterization of these two gonococcal strains isolated from the urethra of MSM to further understand if they are the same strains previously associated with extensive multidrug resistance in other parts of the world.
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    Assessment of total and mobile heavy metals in amended compost of water hyacinth obtained from Lake Victoria, Kisumu County
    (Kenyatta University, 2015-11) Towett, Kiprono Gideon
    Water hyacinth (Eichhornia Crassipes) invasion in Lake Victoria has become a matter of concern over the last two decades. There has been suggestion on the possibility of producing compost from water hyacinth as a way of managing the weed cover in the lake and at the same time help local farmers in the region boost their agricultural production by supplementing the use of commercial chemical fertilizers. However, the safety of using water hyacinth’s compost has to be investigated as studies have shown that water hyacinth accumulates heavy metals from the environment. The specific objectives of the study were to determine the total concentrations of heavy metals (Pb, Cd, Ni and Cu) in water hyacinth compost, to determine the effect of the carbonaceous materials (cattle manure and molasses), and effective microorganisms treatment of water hyacinth’s compost feedstock materials on the mobile concentrations of heavy metals and to determine the relationship between total and mobile concentrations of heavy metals in compost. In the study, water hyacinth compost was prepared using commercial effective microorganisms (EM) solution and carbonaceous materials (cattle manure and molasses) separately as starter cultures for composting. Compost samples were digested with concentrated nitric acid to extract total metals and mobile concentrations were extracted using de-ionized water. Concentration of heavy metals in the compost was determined using Atomic Absorption Spectroscopy and the data collected was analyzed for significant differences (p < 0.05) using one-way Analysis of Variance (ANOVA) with Statistical Analysis System (SAS) Version 9.2 software. Means were separated using Tukey`s test at 5 % level. Pearson correlation analysis was also carried out to check the significances of the linear relations between total and mobile concentrations. Assessment of heat released during composting of water hyacinth showed that water hyacinth compost attained stability and thus maturity after 60 days of composting. The range of total heavy metals in compost samples were 1.23-1.46 mg/kg (copper), 0.32-0.35 mg/kg (cadmium), 0.25-0.32 mg/kg (nickel), 0.95-1.41 mg/kg (lead). The mean concentrations of the mobile heavy metals in mg/kg for the control, EM, manure and molasses treatments were copper: 0.054, 0.055, 0.027 and 0.049. Cadmium: 0.023, 0.032, 0.018, and 0.035. Nickel: 0.036, 0.027, 0.033, and 0.031. Lead: 0.043, 0.061, 0.023 and 0.093. There were significant differences in the mean concentrations of total and mobile concentrations of heavy metals between treatments. The total concentrations of heavy metals in water hyacinth compost, irrespective of treatments, were within acceptable limits and the addition of carbonaceous materials significantly affected the mobility of heavy metals. Nickel had an all positive relationship between total and mobile concentrations. However, for Cu, Cd and Pb an increase in total concentrations did not necessarily correspond to an increase in mobile concentrations. The mobile concentrations of heavy metals in compost were not predictable from their total content. Manure treatment had the least fraction of mobile heavy metals. It is therefore recommended that the composting of water hyacinth for organic fertilizer and as a way of disposing harvested water hyacinth biomass be done. The study also recommends amending of water hyacinth compost feedstock with cattle manure in order to reduce mobility of heavy metals in compost and consequently help alleviate possible adverse effects of compost on the environment.
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    Bacterial and rota viral causes of diarrhoea in children under 5 years and the genetic basis of antibiotic resistance
    (2005) Ndung’u, Perpetual Wangui
    Diarrhoea causes significant morbidity and mortality among children in Kenya. The Illness is caused by a variety of pathogenic organisms, some which are difficult to identify immediately. This results in the use of enormous amounts of antibiotics and as a result, some of the pathogens develop resistance. This study was carried out between June 2003 and January 2004 to investigate the main causes of diarrhoea in children below the age of five years. It also sought to evaluate the problem of antibiotic resistance among these isolates, the role of plasm ids in the resistance and how much of this resistance was transferable. A total of 402 sample stool samples were collected from Kenyatta National Hospital, examined macroscopically to determine the presence of blood, pus, or mucus \\ luch are an important lead in determining the causative agents. The specimens were cultured in general and specific media to isolate different bacteria. Biochemical tests were carried out on suspect colonies for proper identification followed by API 20E screening test. A total of 13 different genera of bacteria were isolated some of which were non-pathogenic. The three genera of pathogenic bacteria isolated including 389 /:".co/i, I 1 Salmonella spp., and 5 ,\'!7iy;,-,IIa spp. Two viral species, 8 rota vi ruses and 2 corona viruses, were isolated from the first 100 specimens. Antibiotic sensitivity tests [vcete carried out on the pathogenic isolates using the Kirby Bauer disc diffu ion method. ".".coli ATCC 25922 was used as control to test the potency of the antibiotics. A total of 12 commonly used antibiotics for treatment of Gram-negative bacteria were used for the sensitivity tests. Plasmid fingerprinting was carried out using Horizontal Agarose Gel Electrophoresis (HEG) on the Ii.coli isolates. In vitro conjugation experiments were also undertaken to determine possible transfer of resistance to Ecoli K 12 F- Na" that has no plasm ids. Results from sensitivity tests indicated multidrug resistance on all three pathogenic strains. The highest resistance was shown by E coli, where four isolates were resistant to II of the 12 antibiotics used. The order of resistance by E.coli was highest on Trill1ethoprim-sulphamethaxazole (89%), and lowest on ceftazidime (4.6%). Salmonella and Shigella showed similar resistance, with highest resistance shown on Trimethoprimsulphamethcprirn (906%), while nalidixic acid, ciprofloxacin and gentamicin were effective on all the isolates of bacterial pathogens isolated. In addition,· all Shigella isolates were sensitive to ceftazidime. The drug resistant Ecoli had plasmids ranging between 2.0 and 98 megadaltons (Mda). Conjugation experiments showed that the ability to transfer resistance phenotypes by the larger plasmids existed for ampicillin, ch loramphenicol and tetracycline. The presence of ciprofloxacin resistant E.coli raises serious concerns and hence need for its frequent monitoring. These results indicate that there is need for constant surveillance on all antibiotics used for treatment of diarrhoea in children and to sensitize the public on the proper use of antibiotics.
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    Characterisation of natural immune responses to fal vac-l of plasmodium falciparumin children and adults from a holoendemic area of Western Kenya
    (Kenyatta University, 2000) Were, Tom
    Previous studies indicate that FAL VAC-1, a recombinant multistage and multicomponent Plasmodium falciparum candidate vaccine containing 12 B cell and 9 T cell epitopes from 9 different antigens of different life cycle stages is immunogenic in animal models and that FAL VAC-1-induced antibodies produced significant antiparasite activities against both sporozoite and blood stages of the parasite. In preparation for vaccine trials in humans, a community based cross-sectional study in a malaria holoendemic area of western Kenya was conducted during April-August 1999, to characterise in vitro natural humoral and cellular immune responses to this candidate vaccine and their association with clinical protection against malaria in young children < 2 years old (N= 180) and their non-pregnant mothers aged 15-48 years (N=139). FAL VAC-1 antigen was used in antibody measurement by ELISA and in lymphoproliferative experiments. Prevalence and level of antibodies were significantly higher in adults than in children when stratified by age groups: 0-6 months; 7-12 months, 13-18 months; 19-24 months; 15-25 years and 26-48 years. Total IgG, IgG1, IgG3 and IgM were the predominant antibodies. IgG2 responses were low and no IgG4 was detected. In children, there were higher IgG 1 levels in parasitaemic group than in the aparasitaemic 'group (F=3.459, p=0.024, t-test). Furthermore, total IgG, IgG1, IgG3 and IgM levels were inversely associated with .' . r '. haemoglobin levels at the time of sampling (total IgG, r=-0.215, p=0.005, IgG1, r=- 0.180, p=0.019; IgG3, r=-0.164, p=0.034; IgM, r=-0.216, p=O.OOl). Parasitaemic children had significantly higher IgG 1 levels at a month prior to sampling. In addition, IgG 1 was positively correlated to the rate of high density parasitaemia and to episodes of clinical malaria (r=0.218, p=0.029 and r=0.237, p=0.018), respectively. However, in adults, aparasitaemic individuals had high total IgG, IgG 1 and IgM levels than parasitaemic individuals (total IgG, F=3.856, p=O.007; IgGl, F=2.701, p=O.007; IgM, F=5.133, p=O.OOl,t-test). In addition, IgG2 was inversely associated with haemoglobin levels at the time of sampling. In contrast to antibody responses, lymphoproliferative responses were higher in children than in adults (one-way ANOV A, F=2.392, p=O.038). Generally, lymphocytes from adults responded at lower antigen concentrations, while those from children responded at higher antigen concentrations. There were no associations between lymphoproliferation and malaria infection or haemoglobin level in either young children or adults. The results of this study therefore indicate that: FAL VAC-l, a multistage multicomponent malaria vaccine candidate is recognised by individuals naturally exposed to malaria. The antibody responses increase whereas lymphoproliferative responses decrease with age. The higher IgG1 levels in children may indicate the presence of a current P. falciparum infection, but in adults from the same holoendemic area, IgG 1, may be associated with protection against parasitaemia.
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    Culturing, Characterization and Identification of Candidate Microorganisms in Cattle Ear, Responsible for Producing Volatile Constituents Attractive to the Brown Ear Tick
    (Kenyatta University, 2015-09) Kogo, Kipchumba Kenneth
    East Coast Fever is a disease of cattle which results in high animal mortality with corresponding economic losses to farmers and to the nation. The disease is caused by a protozoan agent whose vector is Rhipicephalus appendiculatus (Brown Ear Tick). The tick feeds dominantly inside the cattle ear. Studies have shown that the adults of R. appendiculatus exhibit „pull-push‟ effect that guides them to their preferred feeding site. This is linked to the presence of ear volatiles which attracts and anal volatiles which repels the tick. However, there is limited information that describes what produces the volatiles within the cattle ear. This study aims at establishing the role of ear microbes to the brown ear tick behavior while locating its preferred feeding site. In a preliminary observation, release of the ticks on the cattle with one ear sprayed with a broad-spectrum antibiotic showed that most ticks were attracted to the untreated ear, suggesting that the attractant volatiles are derived from specific microbial populations. In the present study where twelve cattle were used, one ear in each cow was treated with vetmycin a broad-spectrum antibiotic while the other ear was left untreated and the orientation behavior of the ticks (R. appendiculatus) placed at six different parts of cattle monitored. Most of the ticks moved to the untreated cattle ear (P<0.001,t-test). Ear swabs were collected and cultured in a general media to obtain a culture of the microbes for in-vitro behavioral tick experiments and genomic studies. In a choice laboratory experiment, ticks were also observed to orient towards cultivable cattle ear microbes relative to a negative control (P< 0.001,t-test). General lab media (Nutrient Agar) was then used to culture different micro-organisms. Sensitivity tests carried out to determine the efficacy of the antibiotic spray used on the cattle ears showed that most microbes were sensitive to the antibiotics contained in the spray according to Kirby Buer calibrations. Profiles of aerobically cultivable microbial communities in each of the twelve cows were investigated using pyrosequencing based analysis. Genomic DNA was extracted and amplified using two sets of primers U1F, U1R which targets band lengths of 490bp and 500bp and primers 27F, 1492R which targets bands lengths of 1490bp. Gene sequences of each ear helped to identify the specific genus of each microorganism and profiles of the candidate microorganisms. Pyro-sequencing blast results revealed that there were diverse communities of microbes majority of which were bacteria. The study lays down significant groundwork in identifying the specific ear microbes responsible for the production of the constituents of ear blend that is responsible for attracting the adults of the Brown Ear Tick. This may open up novel ways of manipulating the production of the blend and in controlling the tick
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    Detection of Pathogenic Human Adenoviruses and Enteroviruses in Water Samples Collected from Lake Victoria along Homa Bay Town, Homa Bay County, Kenya
    (Kenyatta University, 2019-07) Wasonga, Michael Opere
    Lake Victoria is the main source of water in Homa Bay town as well as the surrounding community. Increase in population in the resent past has led to intensified human activities with a possible compromise on the sanitation standards around the town consequently resulting into increased fecal load to the lake through fecal pollution. Increased fecal contamination of the lake has consequently led to an increase in pathogenic microorganisms including waterborne enteric viruses. These viruses can affect both human and animals health by causing diseases such as gastrointestinal infections. Research on viral water quality in Lake Victoria is limited. The objectives of this study were to determine the occurrence of these pathogenic enteric viruses with respect to human adenoviruses and enteroviruses in Lake Victoria Waters. Factors that may have influenced the levels of contamination of the lake water by the viruses such as physical water parameters, seasonal variations, proximity to sewage effluent and pit latrines were assessed. Water samples were collected from six sites commonly used for domestic and commercial purposes spanning approximately 3 km along the shoreline for analysis for the presence or absence of the two groups of enteric viruses over a seven month period. A total of 216 water samples were analysed for possible contamination with the viruses using nested PCR method. Paired t test, ANOVA, Odds Ratios, Correlation and Regression analysis (STATA ver.13.0) were performed to determine factors associated with the virus contamination of the waters. P-values<0.05 were considered significant at 95 % confidence interval. Analysis showed that the lake is contaminated with adenoviruses and enteroviruses which were discovered in 11(5.09 %) and 7(3.24 %) of the samples, respectively. The presence of the enteric viruses was strongly associated with the distance from possible sources of contamination (odds ratio 20.28 and 4.86, confidence interval 2.42, and 0.95) for pit latrines and the sewage treatment plant respectively. Neither wet season nor dry season was associated with the prevalence of the viruses. Of the 72 samples collected from the sites (L5 and L6) closer to the sewage effluent points 13(18 %) tested positive for the two types of viruses. This research clearly signifies that waste water discharge and wastewater-impacted surface waters along the shores of Lake Victoria contain some enteric viruses. This contamination may be due to the fact that the enteric viruses can highly persist in the environmental waters due to their ability to be resistant to environmental conditions. This viral analysis will provide the much needed information in controlling the source of pollution of the lake, such as untreated sewage effluents. It will be useful in ascertaining health risks from the analysis of viral exposure. Further research and analysis is recommended to ascertain the true position concerning the public heath implication and whether the contamination may be as a result of ineffective waste water treatment.
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    Determination of Bacterial Composition, Heavy Metal Contamination and Physico-Chemical Parameters of Fish Pond Water in Abothuguchi Central, Meru County, Kenya
    (Kenyatta University, 2015) Laibu, P. K.
    Fish farming has been practised all over the world for centuries. Throughout history, humans have used fish as a supplementary source of proteins and as a source of income. Fish performs all their body functions in water and therefore the quality of water is very important to their livelihood. Farmers have suffered losses due to the death and stunted growth of fish as a result of bacterial infections and diseases among other causes. The presence of these bacteria in fish pond water and their pathogenic effects varies with the quality of the pond water together with the variation in the frequency with which water is changed in the ponds. Physico-chemical parameters of pond water and heavy metals affect growth and productivity of fish. This research aimed at determining the contamination levels of both faecal and pathogenic bacteria namely Salmonella spp., faecal Streptococcus, faecal coliforms Pseudomonas spp.,Vibrio cholerae and E. coli in fish ponds water, levels of heavy metal contamination and variability of physico-chemical parameters and their effects on fish. Isolation of faecal indicators and pathogens was carried out using standard laboratory methods. Some physico-chemical parameters were measured in situ using a portable Universal multiline P4 WTW (Wilheim Germany) meter while others were analysed in the laboratory. The determination of heavy metal presence and concentration in the water samples was carried out by the use of Flame Atomic Absorption Spectrometer (FAAS). The results indicated that the pond water was highly contaminated with faecal streptococci and faecal coliforms and they varied significantly in the sites at p=0.0001. Pearson correlation analysis showed positive correlation between prevalence of faecal streptococci and faecal coliforms (r=0.732 at P< 0.01). Potential pathogens namely Vibrio spp., Salmonella spp., P. aeruginosa and E.coli were isolated from the water samples with high population. Physico-chemical parameters namel pH and dissolved oxygen deviated from the permissible limits. The study has indicated that the fish ponds water was highly contaminated with both faecal and pathogenic bacteria with physico-chemical parameters varying significantly at p=0.0001. Heavy metals with the exception of iron were within the recommended limits hence no significant contamination of the fish pond water. The study recommends use of treated tap water, regular monitoring of fish pond water and sensitization of farmers on bacterial contamination of pond water. Further studies with the aid of molecular techniques should be used to characterize the bacteria. The finding of this study can, therefore, serve as an impetus to improve fish farming in Meru County, as a way of meeting the growing nutritional demands in the country.
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    Determination of Bacterial Composition, Heavy Metal Pollution and Physicochemical Parameters of Fish Pond Water in Abothuguchi Central, Meru County, Kenya
    (2018-11) LAIBU, PETER KABIRO; MAINGI, JOHN; KEBIRA, ANTHONY
    Laibu PK, Maingi J, Kebira A. 2018. Determination of bacterial composition, heavy metal contamination and physicochemical parameters of fish pond water in Abothuguchi Central, Meru County, Kenya. Bioteknologi 15: 66-79. Humans have consumed fish as a supplementary source of proteins and as a source of income. Because fish performs all their body functions in water, the quality of water is essential to their livelihood. Fish farmers have encountered losses due to the death and stunted growth of fish due to bacterial infections and diseases among other causes. Bacteria found in fish pond water and their pathogenic effects differ with the quality of the pond water along with the variation in the frequency with which water is changed in the ponds. Physico-chemical parameters of pond water and heavy metals influence the growth and productivity of fish. This study aimed at determining the contamination levels of both fecal and pathogenic bacteria namely Salmonella spp., fecal Streptococcus, fecal coliforms Pseudomonas spp., Vibrio cholerae and E. coli in fish ponds water, the concentration of heavy metal contamination and variability of physicochemical parameters and their effects on fish. Isolation of fecal indicators and pathogens was carried out using standard laboratory methods. Some physicochemical parameters were measured in situ using a portable Universal multiline P4 WTW meter while others were analyzed in the laboratory. The determination of the heavy metal presence and concentration in the water samples was carried out by employing of Flame Atomic Absorption Spectrometer. The results indicated that the pond water was heavily contaminated with fecal streptococci and fecal coliforms and they varied significantly in the sites. Pearson correlation analysis showed a positive correlation between the prevalence of fecal streptococci and fecal coliforms. Potential pathogens such as Vibrio spp., Salmonella spp., P. aeruginosa, and E.coli were taken from the water samples with high population. Physico-chemical parameters namely pH and dissolved oxygen deviated from the permissible limits according to international standard. The study has shown that the fish ponds water was highly contaminated with both fecal and pathogenic bacteria with physicochemical parameters varying significantly. Heavy metals except for iron were within the recommended limits hence no significant contamination of the fish pond water. The study suggests the use of treated tap water, routine monitoring of fish pond water and sensitization of farmers on bacterial contamination of pond water. More studies with the aid of molecular techniques should be employed to characterize the bacteria. The finding of this study can, thereby, serve as an impetus to improve fish farming in Meru County, as a way of meeting the growing nutritional demands in the country.
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    Determination of Biodiversity and Symbiotic Efficiency of Native Rhizobia Isolated from Different Regions in Kenya
    (Kenyatta University, 2020-10) Jalloh, Abdul A.
    Crop production has continued to decline in sub-Saharan Africa due to soil infertility and increased cost of farm inputs. To enhance food security, farmers have adopted the use of both inorganic and organic fertilizers on their farms. The production and use of inorganic chemical fertilizers are not only expensive for family farming systems but also contribute significantly to environmental pollution. Biological nitrogen fixation using rhizobia has proven to be a cost-effective and environmentally friendly alternative to inorganic nitrogen fertilizers. Rhizobia interact with legumes symbiotically, improving soil fertility and legume plants productivity. The present study aimed at determining the morphological and genetic diversity, cross-inoculation ability, and symbiotic efficiency of native rhizobia isolates in sterile and non-sterile soils. Greenhouse experiments were set to trap rhizobia from soil from smallholder farms from Kitui, Embu, and Tharaka Nithi Counties using cowpea as the trap host. The cowpea crops were harvested after one month, and a total of 311 nodule isolates were purified from the crop nodules. Based on morphological characteristics, the nodule isolates were clustered into 42 different groups. The effect of the soil on cowpeas nodulation was determined using redundancy analysis where soil characteristics including P, Zn, Mn, and total organic carbon correlated positively with cowpea nodulation and nodule dry weight. Soil pH and Ca correlated negatively with nodule number and weight. In addition, 53 glycerol stocks nodule isolates (archived samples) from previous studies were revived. Revived cultures were clustered based on morphological characteristics into 11 different groups. The isolates were tested for symbiotic efficiency using common bean, cowpea, green gram and soyabean seeds planted in sterile and non-sterile soils in the greenhouse. Un-inoculated plants were used as controls, while the treatments included the revived native isolates and commercial rhizobia inocula. The experiments were laid out in a completely randomized design. Plants grown in both sterilized and non-sterilized soils and inoculated with the different isolates varied significantly (p<0.05) in the shoot, root, and nodule dry weights. Some of the isolates, including; IsAS14, IsAS11, IsAS10, IsAMR6, IsAMR22, IsAMR23, IsAMR27, and IsAGR5 significantly (P<0.05) outperformed commercial isolates in influencing the plants growth parameters. Forty-six revived nodule isolates had the ability to infect, induce nodule formation and influence the growth of the non-original host. The best performing native rhizobia isolate IsAS14, IsAMR3, IsAMR27, IsAMR18 and IsAMR22 outperformed the commercial inocula in terms of symbiotic efficiency of 104.97 %, 136.86 %, 136.99 %, 138.88 % and 155.05 %. DNA from representative isolates was extracted using ZYMO research DNATM extraction Kit. The 16S rRNA region was then amplified using universal primers and sequenced using the same primers. Based on the sequencing of 16S rDNA of representative revived nodule isolates, most of the isolates were rhizobia but clustered with different species with the most dominant cluster being isolates related to Rhizobium leguminosarum and Rhizobium etli. Nodule number and nodule dry weight were dependent on soil physico-chemical characteristics. Rhizobia isolates also had the ability to form symbiotic interaction with non-original host legume crops. These rhizobia isolates can be used as bio-inoculants to improve the production of the different legumes, and to enhance food security.
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    Effect of Soil Amendment With Manure on Nodulation, Biomass and Yield of Soybeans and Climbing Beans in Meru County, Kenya
    (Kenyatta University, 2015) Kithira, I. J.
    The consequence of intense permanent cultivation of land has been severe loss of soil nutrients through removal of harvested produce, removal of crop residue, leaching and gaseous losses. Experiments were designed to investigate the effect of soil amendment with cow manure, chicken manure and fertilizer on nodulation and nitrogen fixation in climbing beans (MAC 64) and soybeans (Gazelle). Isolation and characterization of isolates together with greenhouse experiments were carried out at Kenyatta University laboratory and greenhouse respectively. Field experiments were carried out at Kaguru Farmers Training Centre and Munithu Girls Secondary School farm. The experimental design was a randomized complete block design with four replications. Each of the legume was planted in soils amended with cow manure, chicken manure, fertilizer and other soils left unamended for control. Data was obtained at 75 % podding and full physiological maturity. Laboratory experiments were carried to isolate and characterize the rhizobia nodulating and fixing nitrogen with climbing beans and soybeans. This was carried out using congo red, litmus milk, Bromothymol blue and peptone agar. Greenhouse experiments were carried out to authenticate the rhizobia obtained from the field. Inocula was prepared from the root nodules of each of the legumes. Isolates from climbing beans inoculated radicles of soybeans and those of soybean inoculated climbing beans. Uninoculated climbing beans and soybeans were used as controls. Cross inoculation was also established in the greenhouse. Rhizobia nodulating and fixing nitrogen with soybeans nodulated and fixed nitrogen with climbing beans. Data obtained from the field experiments was analyzed and significance difference determined in nodule number, root dry weight, stover dry weight, pod number and dry weight of 100 seeds. Results were subjected to standard statistical analysis and presented using both descriptive and quantitative statistical procedures. The effect between amendments was determined using Genstat software for the analysis of variance (ANOVA) and mean separated using least significance tests at 5% level. Indigenous bradyrhizobia do not nodulate effectively in soybean and climbing beans in soils with low levels of nitrogen. Nodules did not form in beans planted in the soils amended with nitrogen fertilizer. Cross inoculation of rhizobia was observed between soybean and climbing beans in the greenhouse. Chicken manure, cow manure and nitrogen fertilizer application had effect on nodulation, root dry weight, stover dry weight, pod number and dry weight of 100 seeds on both soybeans and climbing beans. Climbing beans at Munithu Girls secondary School farm showed better performance in soils amended with chicken manure in the parameters assessed. BNF should be exploited with the view of decreasing overdependence on nitrogen fertilizer for sustainable agriculture. There is need for continuous screening of large numbers of native bradyrhizobia to understand their specificity in nodulation and nitrogen fixation
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    Efficacy of some medicinal plants used in various parts of Kenya in treating selected bacterial and fungal pathogens
    (Kenyatta University, 2015) India, Jacqueline
    Medicinal plants have been used since time immemorial to treat and prevent human ailments. WHO indicates that up to 80% of the world's population uses traditional medicine. Infections caused by bacteria and fungi have become a major health problem globally accounting for over 50,000 deaths every day. It is estimated that more than 70% of the pathogenic bacteria are resistant to at least one of the antibiotics commonly used to treat them. Conventional drugs are expensive and have side effects. Many plants have been used by various communities in Kenya in the treatment of bacterial and fungal infections but they have not been validated. The main aim of this study was to determine the efficacy of some medicinal plants used by various communities in Kenya that treat the selected bacterial and the selected fungal diseases in man. An ethnobotanical survey was used to select and collect plants from Mwingi North, Kisii South and Rarieda Districts based on their use to treat infectious diseases such as skin infection, diarrhea and many others. Crude extracts from Zanthoxylum chalybeum, Boscia angustifolia, Melia volkensii, anthoxylum gilletii, Fuerstia africana, Urtica dioica, Vernonia amygdalina, Ricinus communis, Commiphora africana, Psidia puntulata, Senna didymobotrya, Ormocarpum trichocarpum, Sesbania sesban, Balanites aegyptiaca, Albizia coriaria, Ficus sycomorus, Rhus natalensis and Tamarindus indica believed to contain secondary metabolites were screened against ten microorganisms, including the bacteria:- Salmonella typhi ATCC 19430, Escherichia coli ATCC 25922, Bacillus subtilis, taphylococcus aureus ATCC 25923 and Methicillin Resistant Staphylococcus aureus. The fungal strains that were used are; Aspergillus niger, Candida albicans ATCC 90028, Microsporum gypseum, Crytococcus neoformans ATCC 18310 and Trichophyton mentagrophyte. The plants were screened using Kirby Bauer disc diffusion method. Phytochemical screening was carried out to identify the presence or absence of classes of bioactive compounds. Data was analyzed using one way ANOV A, significant means were separated using Tukey's test. Generally, Fuerstia africana, Zanthoxylum chalybeum, Balanites aegyptiaca, Ormocarpum trichocarpum, Senna didymobotrya and Tamarindus indica gave strong antibacterial results of between 14.5 mm and 20 mm as Albizia coriaria, Ficus sycomorus, Commiphora africana Rhus, natalensis Senna didymobotrya, Psidia puntulata, and Tamarindus indica produced strong antifungal results of between 15.5 mm and 20.5 mm. The results ofMICs and the MBCslMFCs of the extracts of Albizia coriaria, Ficus sycomorus, Senna didymobotrya, Psidia puntulata, Fuerstia africana, Balanites aegyptiaca and Tamarindus indica showed a good activity of 0.9375 mg/ml in some test cultures. Salmonella typhi ATCC 19430 and Escherichia coli ATCC 25922 were the least sensitive bacteria while Candida albicans ATCC 90028 was the least sensitive fungus. The present study indicates that the majority of the plantstested are an important source of antibacterial agents especially on Gram positive bacteria(Staphylococcus aureus, Bacillus subtilis and Methicillin Resistant Staphylococcus aureus) and antifungal agents against the dermatophytes especially Microsporum gypseum. This study recommends that the plant extracts with good antimicrobial activitybe subjected to both pharmacological an toxicological studies.
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    Efficacy of some medicinal plants used in various parts of Kenya in treating selected bacterial and fungal pathogens
    (Kenyatta University, 2015-01) Jacqueline, India
    Medicinal plants have been used since time immemorial to treat and prevent human ailments. WHO indicates that up to 80% of the world’s population uses traditional medicine. Infections caused by bacteria and fungi have become a major health problem globally accounting for over 50,000 deaths every day. It is estimated that more than 70% of the pathogenic bacteria are resistant to at least one of the antibiotics commonly used to treat them. Conventional drugs are expensive and have side effects. Many plants have been used by various communities in Kenya in the treatment of bacterial and fungal infections but they have not been validated. The main aim of this study was to determine the efficacy of some medicinal plants used by various communities in Kenya that treat the selected bacterial and the selected fungal diseases in man. An ethnobotanical survey was used to select and collect plants from Mwingi North, Kisii South and Rarieda Districts based on their use to treat infectious diseases such as skin infection, diarrhea and many others. Crude extracts from Zanthoxylum chalybeum, Boscia angustifolia, Melia volkensii, Zanthoxylum gilletii, Fuerstia africana, Urtica dioica, Vernonia amygdalina, Ricinus communis, Commiphora africana, Psidia puntulata, Senna didymobotrya, Ormocarpum trichocarpum, Sesbania sesban, Balanites aegyptiaca, Albizia coriaria, Ficus sycomorus, Rhus natalensis and Tamarindus indica believed to contain secondary metabolites were screened against ten microorganisms, including the bacteria:- Salmonella typhi ATCC 19430, Escherichia coli ATCC 25922, Bacillus subtilis, Staphylococcus aureus ATCC 25923 and Methicillin Resistant Staphylococcus aureus. The fungal strains that were used are; Aspergillus niger, Candida albicans ATCC 90028, Microsporum gypseum, Crytococcus neoformans ATCC 18310 and Trichophyton mentagrophyte. The plants were screened using Kirby Bauer disc diffusion method. Phytochemical screening was carried out to identify the presence or absence of classes of bioactive compounds. Data was analyzed using one way ANOVA, significant means were separated using Tukey’s test. Generally, Fuerstia africana, Zanthoxylum chalybeum, Balanites aegyptiaca, Ormocarpum trichocarpum, Senna didymobotrya and Tamarindus indica gave strong antibacterial results of between 14.5 mm and 20 mm as Albizia coriaria, Ficus sycomorus, Commiphora africana Rhus, natalensis Senna didymobotrya, Psidia puntulata, and Tamarindus indica produced strong antifungal results of between 15.5 mm and 20.5 mm. The results of MICs and the MBCs/MFCs of the extracts of Albizia coriaria, Ficus sycomorus, Senna didymobotrya, Psidia puntulata, Fuerstia africana, Balanites aegyptiaca and Tamarindus indica showed a good activity of 0.9375 mg/ml in some test cultures. Salmonella typhi ATCC 19430 and Escherichia coli ATCC 25922 were the least sensitive bacteria while Candida albicans ATCC 90028 was the least sensitive fungus. The present study indicates that the majority of the plants tested are an important source of antibacterial agents especially on Gram positive bacteria (Staphylococcus aureus, Bacillus subtilis and Methicillin Resistant Staphylococcus aureus) and antifungal agents against the dermatophytes especially Microsporum gypseum. This study recommends that the plant extracts with good antimicrobial activity be subjected to both pharmacological and toxicological studies.
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    Enhancing Cowpea Production through Arbuscular Mycorrhizal Fungi Inoculation and Wide Interspecific Crosses
    (Kenyatta University, 2016-06) Oruru, Marjorie Bonareri
    Cowpea is a multipurpose legume crop that serves as human food, livestock fodder, and income source and is widely produced in sub-Saharan Africa. Soil fertility and attack by insect pests and diseases are significant limitations to its production. Although farm inputs such as phosphate-rich fertilizers and pesticides can solve the mentioned constraints, they are costly for resource-poor farmers. This study aimed at determining the effect of arbuscular mycorrhizal fungi inoculation on AMF root colonization and growth of cowpea. Additionally, it aimed at developing an insect-pest resistant hybrid by crossing the cultivated cowpea (Vigna unguiculata) with a wild (Vigna vexillata) accession. Pot experiments were set up in a completely randomized design using a wild cowpea species (Ni935) and three cultivated cultivars (Katumani 80, Kunde Mboga and KenKunde 1). There were two treatments; mycorrhizal treatment using a commercial inoculum comprising of four mycorrhizal species, Rhizophagus irregularis, Funneliformis mosseae, Glomus aggregatum and Glomus etunicatum and non-mycorrhizal treatment (control). The pots were maintained in a greenhouse for 30 days prior to harvesting. Data on percentage root mycorrhizal colonization, root and shoot dry weights, nodule number and nodule dry weight were recorded. The dried shoots were also analyzed for N, P and K content. Hybridization experiment was done by crossing the cultivated cowpea cultivar (sp 219) with five different wild accessions (Ni935, Ni936, 263, V268 and AC305) to form immature hybrid pods. Data on pod retention frequencies was recorded. All the data collected from the greenhouse experiment was tested for homogeneity of variance then analyzed by two-way ANOVA and Pearson correlation. Results showed a statistically significant effect of genotype and AMF inoculation on percentage root AMF colonization (p<0.001) as well as on other growth parameters. The cultivated cultivars were more susceptible to AMF colonization and had higher root and shoot dry matter content and nodulation compared to the wild species. There was a positive correlation between AMF colonization and the levels of shoot P and N. Shoot P and N nutrition was also higher in the cultivated cultivars than the wild species. Hybridization experiment revealed differences in pod retention among different accessions of Vigna vexillata. However, an insect-pest resistant hybrid was not generated, possibly due to post-zygotic barriers related to interspecific crossing. This study has demonstrated the importance of AMF inoculation in improving cowpea’s performance. Moreover, it has shown that the cultivated cultivars are still more responsive to mycorrhizal inoculation than the wild species. This is contrary to previous studies that have shown that modern breeding programs may result to suppression of mycorrhizal colonization. Therefore, there is the need to screen different cowpea cultivars and other crops for mycorrhizal symbiosis.
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    Evolutionary relationship between Trypanosoma evansi and trypanosoma brucei with respect to specific mitochodrial antigen and phenotype knockout analysis
    (Kenyatta University, 2005) Obanda, Benear Apollo
    Trypanosomiasis is a disease caused by parasitic protozoans of the genus Trypanosoma. The agents of the disease are obligate extracellular parasites that occur in blood, cerebrospinal fluid and tissue fluids. Trypanosoma brucei causes sleeping sickness and agana in sub-Saharan Africa. Trypanosoma evansi causing surra is endemic in Asia, Middle East northern Africa including Northern Eastern Kenya. Salivarian trypanosomiasis is one of the most important and widespread diseases of domestic animals and man in the world. The causes of the re-emergence of this disease include widespread civil war, declining economies, reduced health financing and the dismantling of disease control programs. The current drugs in use are toxic and not effective because of drug resistance, hence, the need for developing new drugs. The study objective was to establish the evolutionary relationship between T brucei and Tevansi, with respect to cell differentiation life cycle specific antigens and phenotype knockout analysis. PCR was used to compare genes encoding mitochodrial protein of Tevansi IL 1695, Tevansi IL 1934 and Trypanosoma brucei rhodesiense IL2343. Plasmid construction, preparation of plasmid DNA was done using alkaline lysis method. Extraction and purification of plasmid was by QIAGENR plasmid protocol. Cell line of T evansi and T brucei for RNA interference experiments were established. Electroporation was by Gene-Pulse machine for generation of knockout phenotypes. Statistical analysis was by Student's t-test. Tevansi IL1695, Tevansi III934 and T b. rhodesiense IL2343 contain all the five genes for mitochodrial protein in their genomes. MP 48 and MP 52 RNA editing ligases genes were identified in Trypanosoma evansi. Specific RNA ligases MP 48 630 bp and MP 52 560bp primers were developed. These primers specifically identify T evansi, T brucei and T equiperdum from other organisms. Alignment of MP 48 and MP 52 gene sequences obtained in T evansi and T brucei show 100 % homology. Comparisons of MP 48 and MP 52 RNA ligase gene with data of closely related organisms available in Genbank® showed no significant homology with the RNA ligase sequences of TcruziREL and L. majorREL2 sp. nor with the available sequences ofLt RNA ligase. Multiple alignment of T evansi MP52 and MP 48 with related proteins show a perfectmatch with T brucei and near-perfect match of genes with data of closely related organisms available in Genbank. T evansi was able to use T7 promoter gene, to recognize bacteriophage T7 RNA polymerase and produce RNA polymerase that synthesize mRNA encoding Green fluorescent protein, that was observed as Green fluorescent Tievansi. Approximately 40% of original populations of both the species were killed due to RNA interference. There was no significant difference in the effect of RNA interference in Tbrucei Gutat 3.1 and 'Levansi Tansui 13, using Student's t-test twotailed, p> 0.05 at 95% confidence interval. These new sub genus specific primers can be used as a diagnostic tool for monitoring pathogenic Trypanozoon parasites in humans, domestic animal. The primers can also be used in epidemiological survey. The RNA interference analysis identified MP48 and MP 52 RNA editing ligases as a drug target for the development of novel therapeutics in treatment of sleeping sickness, nagana and surra.
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    Genetic Diversity and Antibiotic Resistance of Escherichia Coli Isolated from Human, Cattle, and Buffalo in the Maasai Mara Ecosystem, Kenya
    (Kenyatta University, 2022) Onyari, Winfridah Bwari; Ezekiel Mugendi Njiru; Vincent Obanda
    Antibiotic resistance is an emerging health crisis globally with a significant impact on human and animal populations. The emergence and spread of antibiotic resistance are attributed to the wrong use of antibiotic products in human and animal health care. This is further amplified and complicated in the human-livestock-wildlife interfaces where there is selective pressure and close interaction. This interface and its impact on antibiotic resistance dynamics are inadequately evaluated in Kenya. The study aimed to establish the genetic diversity and prevalence of antibiotic resistance of Escherichia coli isolates from human, cattle, and buffalo populations in the Maasai Mara Ecosystem located in Kenya. E. coli was isolated by phenotypic and biochemical methods from stool samples of humans and fresh dung samples of cattle and buffalo collected from the Maasai Mara Ecosystem. Molecular techniques were used to characterize E. coli isolates. E. coli isolates were tested against tetracycline, gentamicin, ciprofloxacin, ceftriaxone, and amoxicillin-clavulanic acid using the Kirby-Bauer disk diffusion method and then assigned to phylogenetic groups according to chuA, yjaA, and TspE4.C2 genetic markers. E. coli isolates exhibiting resistant strains were genetically characterized based on their 16S rRNA gene region of the gDNA. Phylogenetic analysis established that all the four phylogroups (A, B1, B2, and D) were present in the E. coli isolates from the sympatric hosts except phylogroup B2 which was absent in buffalo population. E. coli isolates from the sympatric hosts were predominant in phylogroup A and B1. Buffalo was predominated by isolates of E. coli in phylogroup B1 and D. Further, the genetic sequence of resistant E. coli isolates recorded 14 unique haplotypes, with haplotype 4 and haplotype 7 being present in all the three sympatric hosts. Humans recorded the highest resistance to the tested antibiotics at 94% followed by buffalo at 50% and cattle at 45%. Humans, cattle, and buffalo recorded the highest antibiotic resistance to tetracycline at 83%, 45%, and 33%, and lowest antibiotic resistance to amoxicillin-clavulanic acid at 46%, 94%, and 100% respectively. Further, antibiotic resistance was prevalent in isolates of E. coli from buffalo with interactions and the resistant pattern exhibited those of humans and cattle against tetracycline 27%, gentamicin 23%, and ciprofloxacin 14% as compared to E. coli isolate from isolated buffalo which only recorded resistance to tetracycline antibiotic at 5%. It was concluded that isolates of E. coli from humans, cattle, and buffalo were genetically diverse. E. coli isolates from the sympatric hosts were resistant to frequently used antibiotics in human medicine and veterinary practices and finally, antibiotic-resistant in buffalo correspond to those antibiotics used in humans and food-producing animals. It was recommended that additional studies using a One-Health approach, are needed to identify the main reservoir of antibiotic resistant strains and to determine the transmission pathway in the multi-host system.