Theses and Dissertations (Biosciences)

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Assessing the effect of some plant extracts on Fusarium oxysporum f.sp. cubense
(Vaal University of Technology, 2022-12) Tebeila, Mashego Kabelo; Pillay, M., Prof.; Takaidza, S., Dr.
Fusarium oxysporum f.sp.. cubense (FOC) causes fusarium wilt, one of the most destructive diseases of bananas. The management of F. oxysporum through synthetic fungicides causes serious environmental problems and threatens to non-target organisms. Medicinal plants may be a good substitute for synthetic fungicides due to their fewer negative impacts on human and ecological health. This study assessed the antifungal effect of crude extracts of Allium cepa L, Allium sativum L, Curcuma longa L, Tulbaghia violacea Harv, and Zingiber officinale L against FOC. The extracts were prepared using acetone, methanol, and water through maceration. Qualitative phytochemical screening was performed using standard methods. The plant extracts’ total phenolic and tannin content was determined using the Folin Ciocalteu method. Total flavonoids were determined by using the aluminium chloride colourimetric method. The antifungal activity of the extracts was determined by well diffusion, microtiter, and poisoned food techniques. High-Performance Liquid Chromatography (HPLC) profiling of standard phenolic compounds in T. violacea and A. sativum was conducted. Most of the tested phytocompounds were present in the acetone and methanol extracts of C. longa and acetone extract of A. sativum. The total phenol, flavonoid and tannins varied in the different plant extracts ranging from 0.23 to 50.56 mg GAE/g, 0.65 to 17.73 mg QE/g and 2.48 to 129.65 mg TE/g, respectively. The acetone and methanol extracts of T. violacea and A. sativum showed inhibition zones against the FOC (17–26 mm), while the water extract showed no inhibition. The minimum inhibitory concentration of the extracts ranged from 1.56 mg/mL to 50 mg/mL, with T. violacea and A. sativum showing the best activity. The methanol and acetone extracts of T. violacea and acetone extracts of A. sativum fully inhibited the mycelial growth of FOC. HPLC analysis of the extracts with the best antifungal activity revealed the presence of the following phenolic compounds: tannic acid, ascorbic acid, benzoic acid and acetyl acid in the acetone and methanol extracts of T. violacea and acetone extract of A. sativum. This research shows that T. violacea and A. sativum may be used to formulate new, safer, and ecofriendly fungicides for F. oxysporum f. sp cubense. Therefore, plant extracts could be a good alternative in managing fusarium wilt in bananas.
Assessing methanol and water leaf extracts of Eriobotraya japonica (Thunb) Lindl for anti-diabetic properties
(Vaal University of Technology, 2022-11) Sikhakhane, Maria Nomusa; Pillay, Michael, Prof.; Takaidza, Samkeliso, Dr.
Diabetes mellitus is projected to become one of the world’s leading causes of death and disability in the next 25 years. By 2004, diabetes mellitus was widespread, affecting approximately 25% of the world’s population. Despite developing several drugs, diabetes and its secondary complications remain a major health problem. Many biochemical and chemical agents used to treat hyperglycemia have known side effects. Herbal medicine is used as an alternative form of treatment for diabetes in developing countries where the cost of conventional medicine is still high. This research aimed to assess methanol and water extracts of the leaves Eriobotrya japonica for anti-diabetic properties in vitro. The leaf extracts' bioactive molecules were characterized by thin-layer chromatography (TLC) and Liquid chromatography-mass spectrometry (LC-MS) analysis. The total antioxidant capacity and free radical scavenging activity of the aqueous and methanolic-based extracts were assessed using the DPPH assay. The inhibitory effects of the aqueous and methanolic extracts on alpha-amylase and alpha- glucosidase were examined to evaluate the antidiabetic potential. The qualitative phytochemical analysis showed that pharmacologically active compounds such as tannins, terpenoids, flavonoids, steroids, cardiac glycosides, and phenols were present in the leaves of E. japonica. However, alkaloids were absent in both extracts. The quantitative phytochemical analysis revealed that E. japonica has a lower Total Flavonoids Content (TFC) than Total Phenolic Content (TPC), and this trend was observed in both extracts. The water extract had a TFC of 0.085 ± 0.004 mg of QE/g and TPC of 19.88± 0.2 mg of GAE/g, while the methanol extract had a TFC of 0.084 ± 0.02 mg of QE/g and TPC of 19.10± 0.1 mg of GAE/g. A total of 12 bands were observed on the (TLC) plate of the methanol extract, while none was observed for the water extract. From the (LC-MS), 13 compounds were identified from the water extract of E. japonica. Protocatechuic acid occurred in the highest concentration at 115 mg/L, while Icariside F2; Benzyl beta-D-Apiofuranosyl-(1->6)-O-beta-D-glucopyranoside was found in the lowest concentration of 0.1 mg/L. A total of 14 compounds were characterized from the methanol extract of E. japonica. Blumenin, which belongs to a group of fatty acyl glycosides of mono- and disaccharides, had the highest concentration at 82.7 mg/L, whereas xanthohumol had the lowest concentration at 10.5 mg/L. The 2,2-diphenyl-l-picrylhydrazyl-Thin Layer Chromatography bioautography assay showed that the water extract had 5 bands of compounds with antioxidant activity while the methanol extract showed 6 bands. The free radical scavenging assay was performed using DPPH to qualify antioxidants in E. japanica. The ascorvic acid, methanol, and water extracts had the highest percentage inhibition of 85.9%, and 71.6%, and 40.7%, respectively. The IC50 values for ascorbic acid, methanol, and water extracts were 4.4, 6.1, and 12.2 μg/mL, respectively. Alpha-amylase and alpha-glucosidase activity inhibition assays were performed to determine if E. japonica leaf extracts can slow down the rate at which carbohydrates are digested and absorbed into the blood stream, thus reducing blood sugar levels. The highest concentration of 5 mg/mL of the methanol and the water extracts inhibited 88.5% and 83.01% of alpha-amylase activity, respectively, while acarbose inhibition was at 91.3%. The lowest concentration of 0.0391 mg/mL of the methanol and water extracts inhibited alpha-amylase activity by 40.19% and 22.85%, respectively, while acarbose inhibited the activity by 12.4%. The IC50 values of acarbose, methanol and the water extract were 0.07, 0.03, and 0.06 mg/mL. The highest concentration of 5 mg/mL of the water and the methanol extracts inhibited 72.8% and 77.3% of alpha-glucosidase activity, respectively, while acarbose inhibition was 85.1%. The lowest concentration of 0.0391 mg/mL of the water and methanol extracts inhibited alpha-glucosidase activity by 38.9% and 40.7%, respectively, while acarbose inhibition was 32.0%. The IC50 values of acarbose, methanol and the water extract were 0.03, 0.026, and 0.027 mg/mL. The methanol extract was more potent than the water extract in inhibiting alpha-amylase and alpha-glucosidase activity. The findings from the enzyme activity inhibition assays indicated that E. japonica slows down glucose production and absorption in a dose-dependent manner. Based on the results of this study, it can be concluded that the leaf extracts of E. japonica have bioactive compounds, which can be explored for managing type 2 diabetes.
Assessing the effectiveness of the water purification process in removing clostridium perfringens spores as a surrogate for protozoan parasites
(Vaal University of Technology, 2022-12) Schubart, Annah Lindiwe; Swanepoel, Annelie, Dr.; Marrengane, Zinhle; Ssemakalu, Cornelius Cano, Prof.
The World Health Organization (WHO) provides guidelines for assessing the microbiological risk associated with drinking water using a Quantitative Microbial Risk Assessment (QMRA). Microbiological risk in water may arise from pathogens such as protozoan parasites Giardia and Cryptosporidium. The risk presented by Giardia and Cryptosporidium in water is increased by the fact that these pathogens are resistant to disinfection by chlorine. It is costly to monitor treated water for the presence of Cryptosporidium and Giardia, therefore a surrogate was used to carry out the evaluations. This research first determined the efficacy of the conventional water treatment processes in removing Clostridium perfringens spores as a surrogate for protozoan parasites (Cryptosporidium oocysts and Giardia cysts). We estimated the number of protozoan parasites that can pass through the drinking water treatment barriers. This removal efficiency can then estimate the number of protozoan parasites that can survive drinking water purification. The study conducted a simulated jar test under predetermined conditions using three different coagulation combinations: (i) Polyelectrolyte, (ii) polyelectrolyte and slaked lime, and (iii) slaked lime and activated sodium silicate. The three regimens (polyelectrolyte, polyelectrolyte and slaked lime, and slaked lime and activated sodium silicate) were tested each at low temperatures (12.8 ± 0.4°C), normal temperatures (17.2 ± 0.9°C), and at high temperatures (20.3 ± 0.1°C). The three coagulant combinations (polyelectrolyte, polyelectrolyte and slaked lime, and slaked lime and activated sodium silicate) were also tested under normal temperature conditions at high water turbidity. Percentage and log reduction for C. perfringens spores were calculated for each water treatment unit. A percentage reduction for turbidity was calculated for each water treatment unit. Pre-experiments were conducted to determine the suitability of the filter membranes and media to be used for analysing the water samples for the study. The 0.45 μm filter membrane and the Perfringens Agar Base (PAB) were selected and used. Experiments conducted at low temperatures (12.8 ± 0.4°C) showed C. perfringens spore log reductions of infinity or 100% when the polyelectrolyte and a combination of polyelectrolyte and slaked lime were used. Under normal conditions (17.2 ± 0.9°C), C. perfringens spores log reductions of up to 2.0 or 98.9 ± 0.4% were observed using a combination of polyelectrolyte and slaked lime. However, when experiments were conducted at high temperatures (20.3 ± 0.1°C), C. perfringens spore log reductions of infinity or 100% were observed with the polyelectrolyte. At increased turbidity, C. perfringens spores log reductions of up to 2.0 or 99.1 ± 0.1% were observed with the polyelectrolyte. The lowest turbidity reduction of up to 24.1 ± 0.8% was observed when slaked lime and activated sodium silicate were used at 20.3 ± 0.1°C. The highest turbidity reduction of up to 99.0 ± 0.1% was observed using a combination of polyelectrolyte and slaked lime for high-turbidity water. This study showed that water purification steps could remove up to 100% of C. perfringens spores when a polyelectrolyte or a combination of polyelectrolyte and slaked lime are used under varying conditions. Therefore, this study recommends using a polyelectrolyte or a combination of a polyelectrolyte and slaked lime for water treatment under specified conditions. A polyelectrolyte and slaked lime combination is recommended for high-turbidity water.
The antifungal activity and bioanalysis of fractions from Tulbaghia violacea (leaf and root) acetone extracts.
(Vaal University of Technology, 2022-12) Makgati, Mogau Mafise Brian; Mitema, A. Dr.; Takaidza, S. Dr.
Infectious diseases represent a critical problem to health and cause morbidity and mortality worldwide. Despite the significant progress in human medicine, infectious diseases caused by microorganisms such as fungi are still a major threat to public health. Tulbaghia violacea is one medicinal plant that has been used traditionally to manage fungal infections. The present study aimed to determine the antifungal activity and perform bioanalysis of acetone fractions from T. violacea extracts. Tulbaghia violacea crude leaf and root acetone extracts and their fractions were tested against seven fungal species for antifungal activity. The plant extracts were prepared using the maceration method, and column chromatography was utilized to obtain fractions. The plant extract’s total phenolic and flavonoid contents were determined using the Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. The antioxidant activity was evaluated using the free radical scavenging methods 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS•+) assays. The antimicrobial activity of T. violacea crude leaf and root extracts and their fractions was determined using the well diffusion, microtiter, and time-kill assay. The compounds present in the T. violacea leaf and root extracts and their fractions were determined using GC-MS. The results indicated that the total phenolic content (TPC) of all four samples of T. violacea leaf extracts ranged from 0.597-5.003 mg of Gallic acid equivalent/g, whereas total flavonoid content (TFC) varied between 5.324- 15.844 mg of Quercetin/g. The TPC and TFC of all root extracts ranged between 3.19-8.41 mg of GAE/g and 10.28-12.40 844 mg of QE/g, respectively. DPPH leaf fractions ranged from 48-50% and 60-61% for root extracts at 0.5 mg/mL. The ABTS assay also showed a dose-dependent radical scavenging activity. The scavenging activity ranged between 92-95% (crude leaf extract and fractions) and 77-84% (crude root extract and fractions). The investigations using GC-MS revealed a total of 21 metabolites with varied phytochemical activity. For antimicrobial activity, all T. violacea leaf and root acetone extracts and their fractions suppressed the growth of all fungal strains with leaf extracts zones of inhibition ranging between 3-18 mm whereas the root extracts ranged between 3.3-21 mm. The MIC of leaf and root acetone extracts and their fractions ranged between 0.78-12.5 mg/mL and 0.39-12.5 mg/mL, respectively whereas the MFC values of the plant leaf and root acetone extracts and their fractions ranged between 0.78- ˃ 50 mg/mL and 0.39-25 mg/mL, respectively. The time-kill curve assay demonstrated that most of the leaf extract and their fractions at 2 MIC demonstrated fungistatic activity averaging 3-6.23 log10 kill against C. albicans whereas, almost all root extract and their fractions demonstrated fungicidal activity, averaging 0.04-3 log10 kill. The membrane permeability assay indicated membrane damage in a dose-dependent manner for all extracts. In conclusion, T. violacea possesses phytocomponents with good potential as antifungals.
Antimycobacterial activity of synthetic compounds isolated from South African medicinal plants against mycobacterium tuberculosis
(Vaal University of Technology, 2014-11) Ledwaba, Elizabeth Ramadimetsa; Bapela, N. B., Dr.; Van Wyk, Christa
Tuberculosis (TB) remains one of the most difficult infectious diseases to control in the world today. The disease spreads easily in overcrowded, badly ventilated places and among people who are undernourished. Trends in the incidence of TB together with the development of multi-drug (MDR-TB) and extensively drug resistant (XDR-TB) strains of TB raises the need to intensify the search for more efficient drugs to combat this disease. Herbal remedies used in traditional medicine provide an interesting and largely unexplored source for the discovery of potentially new drugs for infections such as TB. The aim of the study was to evaluate the in vitro antimycobacterial activity of synthesized compounds from medicinal plants against Mycobacterium tuberculosis (M. tuberculosis). About 40 synthesized compounds isolated from South African medicinal plants were screened against H37RV using microplate alamar blue assay (MABA). Identified active compounds were screened against resistant strains of M. tuberculosis (MDR, XDR and pre-XDR) and sensitive clinical isolates of TB. Cytotoxicity and synergistic drug combination studies were done on active compounds to validate their toxicity and synergy levels. Cytotoxicity was done by sulforhodamine assay (SRB) against the C2C12 cell line. Only six compounds showed activity against M. tuberculosis with minimum inhibitory concentration (MIC) below 10μg/ml. The results obtained indicated that the cytotoxicity effects of the three compounds on C2C12 cells demonstrated marginal toxicity except for MVB 282/61215 which showed a high toxicity at the lowest concentration of 0.156μg/ml with over 100% viable cells at the highest concentration (5μg/ml). MVB 282/61271 had the highest percentage cell viability (65%) at the lowest concentration. Only two compounds had a higher potency evoking a bigger response at low concentrations with treated cells still viable after 3 days of incubation with the compound which was comparable with the treatment of isoniazid (INH). Synergistic activity of the six compounds was less in INH combination as compared to the rifampicin’s (RIF) combination. The results demonstrated that the synergistic interaction between the compounds and RIF could the antituberculosis acitivity. In conclusion the synergistic effects with RIF translate to lower dosing requirements of the compounds and the potential to combat multidrug resistant TB. In deed there is no doubt that natural products, with their range of interesting chemical structures and powerful antimycobacterial effects are certain to remain important participants in the development of new generations of antimycobacterial drugs.
Investigating the efficacy of a moving bed biofilm reactor for the removal of the antiretrovirals tenofovir, emtricitabine, nevirapine, ritonavir and efavirenz from synthetic wastewater
(Vaal University of Technology, 2022-04) Mokgope, Herman D.; Klink, Michael, Dr.; Walmsley, Tara, Dr.
South Africa utilises more antiretroviral (ARV) compounds per capita than any other nation in the fight against Human Immune Deficiency Virus (HIV) or acquired immunodeficiency syndrome (AIDS). Considering the main entrance pathways of antiviral drugs into the urban water cycle, excretions via urine or faeces from treated individuals play a dominant role. Due to the limited efficiency of conventional biological treatment (activated sludge), ARVs were detected in South African wastewater treatment plant effluents and surface waters. This poses a threat to aquatic environments due to the toxicity of ARVs and can be a potential contributor to ARV resistance due to persistent low level ARV exposure in the general population. This study investigated the efficacy of a moving bed biofilm reactor (MBBR) for ctybtri8nthe elimination of five ARV compounds i.e., tenofovir, emtricitabine, nevirapine, ritonavir and efavirenz from synthetic wastewater. Furthermore, the study also looked at the shift in microbial community compositions of biofilms in the MBBR due to exposure to the ARV compounds. Lastly, the ecotoxicity of the MBBR’s influent and effluent along with the actual ARV compounds were examined. The capacity of ARV degradation by the MBBR was investigated by spiking synthetic wastewater influent with 10 μg/L of five ARV compounds. Actual removal during treatment was assessed by sampling the inlets and outlets of the reactor. A targeted solid phase extraction method with Ultra High Pressure Liquid Chromatography coupled to quadrupole time of flight mass spectrometry (LC-MS/MS) was used to quantify the five ARV compounds. Microbial diversity (alpha-diversity) of seeded sludge from a full-scale municipal WWTP and biofilm samples from a laboratory scale MBBR system during pre- and post-introduction of ARV compounds was investigated by Illumina sequencing of the 16S rRNA gene. Ecological toxicity of the MBBR’s influent and effluent along with the five ARV compounds was determined using the Vibrio fischeri, Daphnia magna and Selenastrum capricornutum toxicity test kits and measured as EC50. After MBBR treatment; Nevirapine, Tenofovir, Efavirenz, Ritonavir and Emtricitabine all showed marked reduction in concentration between the influent and effluent of the MBBR. On average, the percentage removed for Nevirapine, Tenofovir, Efavirenz, Ritonavir and Emtricitabine was 62.31%, 74.18%, 93.62%, 94.18% and 94.87% respectively. Microbial diversity results demonstrated that the introduction of antiretroviral drugs affects the bacterial community composition and diversity considerably. For instance, Nitrosomonas, Nitrospira and Alicycliphilus were found to be higher in post introduction of ARV compounds biofilm samples than in biofilm samples before the introduction of ARV compounds. The EC50 for Tenofovir, Emtricitabine, Nevirapine, Ritonavir and Efavirenz were 82.5, 41.7, 39.3, 60.3 and 0.21 mg/L respectively for S. capricornutum; 81.3, 50.7, 49, 87.1 and 0.43 mg/L respectively for D. magna; and 73.5, 55.1, 41.3, 83.6 and 0.55 mg/L respectively for V. fischeri. The EC50 of the influent and effluent were found to be above 100% concentration, therefore they could not be specifically determined. The ecotoxicity results show that ARV compounds are potentially toxic to the environment, with efavirenz being more toxic than the other four ARV compounds tested. Since there were no toxic effects observed from the effluent, it can be assumed that mineralisation has occurred, or the transformation products are of less or equal toxicity to the influent (because the influent did not show any toxic effects to the model organisms tested).
The effect of a sugar sweetened beverage diet on DNA methylation in a CACO-2 cell line in vitro
(Vaal University of Technology, 2020-12) Ndhlovu, Lesego; Pillay, M., Prof.; Ssemakalu, C., Dr.
Obesity has steadily increased and represents a major public health problem worldwide, reducing quality of life and causing a range of health problems. Obesity has emerged as the fifth leading risk of global deaths. Annually, 2.8 million adults die as a result of being overweight or obese. The increase of obesity remains inexplicable in terms of genetic susceptibility to obesity. The genetic loci identified by genome-wide association studies (GWASs) explains about 2% of the heritability for obesity. Perhaps other factors such as epigenetics may be involved in the increase of obesity and may offer solutions for the management of obesity. Epigenetics is defined as a heritable change in gene expression without altering the genome sequences. It may help in providing a logical explanation between the genome and environment which shapes obesity risk and may help to explain the "missing heritability". Epigenetics may affect two mechanisms, namely: i) DNA methylation,and ii) histone modifications. DNA methylation might give scientists a link to the rise in obesity.The study aimed to investigate the effect of sugars used as sweeteners in sugar-sweetened beverages (SSB) on DNA methylation in a Caco-2 cell line in vitro. Four major objectives were pursued in the study which were to:(1) stimulate the Caco-2 cells with varying concentrations of sugar sweeteners and assess the morphological changes of the cells; (2) evaluate the cytotoxicity of different concentrations of the sugar sweetener on the Caco-2 cell line using the Alamar blue and LDH assay; (3) obtain genomic DNA from the treated Caco-2 cell line and perform bisulfite conversion and rest; and (4) amplify the WT1, MEG3, TNFRSF9, ATP10A, and CD44 obesity-associated genes and ascertain their degree of methylation. Caco-2 cells were stimulated with sugar sweeteners at varying concentrations (low, medium and high) for an incubation period of 62 days,and images of the cells were captured for morphological characterisation. The incubation condition entailed cells plated in a 12 or 96 well plate, incubated in a humidified 5% CO2 incubator at 37 °C and there is nutrient renewal every three days.Alamar blue, a cell proliferation colourimetric assay and lactate dehydrogenase assays (LDH), a homogenous membrane fluorimetric assay were used for the cytotoxicity studies. The results of the characterisation showed that different concentrations of sugar sweeteners affected the morphology of the cells as the incubation period progressed. The cytotoxicity results of both LDH and Alamar blue depicted low concentration of sweeteners that had low-to-moderate toxicity and the medium and high concentration of the sweeteners had a moderate to high toxicity on the Caco-2 cells. DNA from the Caco-2 cells was extracted. Techniques used to study DNA methylation such as bisulfite conversion, PCR amplification and restriction enzymes that have differential sensitivity to 5-methyl-cytosine were performed. The quality of DNA extracted was good. The bisulfite conversion was conducted andno amplification was observed, as a contingency plan Normal PCR was performed to amplify the CpG islands, and there was amplification. In conclusion, the study showed that a low concentration of a sugar sweetener (fructose: glucose) used in beverages had low toxicity to the Caco-2 cell line and prolonged exposure of the low concentration might have an adverse effect on the cells' morphology. At medium concentrations, the sugar sweetener used in beverages had medium toxicity to Caco-2 cells; prolonged exposure may lead to morphological changes. These findings indicated that control of dietary glucose intake is an important strategy in combating the development of obesity and type-2 diabetes. DNA methylation could not be established.
Synthesis, characterisation and assessment of antimicrobial activity of doped zinc oxide nanoparticles against selected waterborne pathogens
(Vaal University of Technology, 2019-07-29) Volofu, Nomasamariya Elsie; Laloo, N.; Mthunzi, F., Dr.; Klink, M., Dr.
The aim of the study is to synthesise, characterize and assess the antimicrobial activity of cobalt oxide, zinc oxide and cobalt-doped zinc oxide nanoparticles against selected waterborne pathogenic fungi (yeasts and moulds) and bacteria. Various types of oxide based nanomaterial are an attractive option for the disinfection of water due to its high chemical stability and non-toxicity towards human cells. Synthesis of Co -doped ZnO and Co3O4nanoparticles was done through mechanochemical synthesis and urea based synthesis and microwave heating was employed for the preparation of ZnO nanoparticles. The ZnO nanoparticles were produced in short reaction and it was white color. Cobalt oxide (Co3O4) nanoparticles appeared as a pink precipitate but was turned black after being calcined. The synthesis of Co- ZnO nanoparticles was successfully prepared and blue solid was obtained from pink cobalt ion solution. The nanoparticles were characterised by X- Ray Diffraction (XRD), Fourier Infrared Spectroscopy (FTIR), UV–visible spectroscopy, Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) (Yang et al. 2003). In this research project, the antibacterial activities of NPs were carried out by well diffusion method and minimum inhibitory concentration (MIC). MIC is the lowest concentration of a chemical, usually a drug, which prevents visible growth of bacterium. Bacterial strains used in the study are: Salmonella enterica, Escherichia coli, Shigella sonnei and Staphylococcus aureus, yeast and mould is: Candida albicans and Aspergillus niger. The antimicrobial results obtained showed that ZnO nanoparticles are more effective than Co- ZnO and Co3O4 nanoprticles against all the microorganisms used. The toxicity studies were performed using DAPHTOXKIT F and the 24h EC50 and 48h EC50 were calculated according to the manufactures’ instructions. The results showed that Co- ZnO nanoparticles is less toxic to Daphnia magna compared to ZnO and Co3O4 NPs.
The prevalence of HBV, HTLV, HIV and concurrent infections in blood recipients of the South African National Blood Service (SANBS)
(Vaal University of Technology, 2020-12) Willemse, Reynier; Vermeulen, M.; Grobler, C. J., Dr.
Background: Currently, the South African National Blood Services are not testing for HTLV and HTLV screening is not mandated by the WHO or by regulatory standards in South Africa. Looking at the uniquely high prevalence of HIV and HIV / HBV co-infections in the South African population and taking into account the literature that suggests that most of these infected patients will be receiving blood, exposing these patients to an additional burden like HTLV can result in an increased disease progression of HIV to AIDS and a poor prognosis in these infected patients. Study design and methods: A blinded cross-sectional study was performed. 7015 specimens were collected from all blood transfusion laboratories across South Africa excluding the Western Cape Blood Transfusion Service laboratories. The specimens collected were tested using the ABBOTT Alinity S® Immunochemiluminescent autoanalyser. All test results were confirmed with the Roche Cobas® E801 and E411 auto analyser. Results: Over all prevalence for HIV was 39.39% (N=2763), HBV 7.57% (n=531) and HTLV 0.70% (N=49). Concurrent infection for HIV/HBV 4.92% (N=345), HIV/HTLV 0.36% (N=25), HBV/HTLV 0.09% (N=6) and HIV/HBV/HTLV 0.07% (N=5). Conclusion: This study confirmed an overall high prevalence of HIV and HBV infections among patients receiving blood products from the SANBS. Compared to the general population, the HIV prevalence in blood recipients was two-fold higher. Patients receiving a blood transfusion from the SANBS have high rates of HIV, HBV and HTLV which should be taken into consideration when determining donor screening strategies.
Assessing the pollutant removal efficiency of a wetland as a polishing treatment for municipal wastewater
(Vaal University of Technology, 2021-02-16) Mphuthi, Betty Refilwe; Osifo, P., Prof.; Walmsley, T. A., Dr.
Pollution of aquatic systems by wastewater containing pathogens, heavy metals and high concentrations of nutrients is of great concern due the ecological risks they impose. The toxic effects of metals may occur even at low concentrations because of potential bio magnification in the food chain. Excessive nutrients cause algal blooms which depletes oxygen and prevents sunlight from penetrating into the water, thereby killing fish and other aquatic organisms. This study investigated the pollutant removal efficiency of a riparian wetland located in Sebokeng, Emfuleni local municipality, South Africa. The study was carried out to assess the water quality of a wetland located downstream of the Sebokeng wastewater treatment plant by monitoring and analysing the physico-chemical parameters which included pH, temperature, electrical conductivity, nutrient levels (nitrates, phosphates, nitrites) and heavy metals. The water samples were collected from the effluent discharge of the treatment plant, upstream and downstream of the wetland. Plant uptake of heavy metals in a riparian wetland, nitrification as well as denitrification processes have been historically recorded as the main processes that contribute to the high removal of pollutants in a wetland. The contaminant concentrations of the influent and the effluent were used to estimate the wetland efficiency in improving the water quality that passes through it and its potential effects on improving the quality of irrigation waters. The heavy metals of interest included Al, Cd, Cr, Cu, Fe, Pb, Mn and Zn. Most heavy metals within the wetland occurred at low concentrations (lower than detectable limits and within the discharge limits for irrigation purposes). The results indicate that the average removal efficiencies for Electrical Conductivity (EC), Total coliforms (TC), E. coli, BOD5, COD, TSS, carbonate hardness, aluminium, iron, manganese, copper, nitrite, nitrate, sulfate and ortho-phosphate were 43 %, 51%, 85%, 60%, 61%, 61%, 21%, 67%, 52%, 51%, 83%, 56%, 89%, 49% and 54% respectively. The study showed that this wetland can provide up to 89% removal efficiency of pollutants. Of particular significance was the high pathogen and nutrient removal efficiency. A t-test was performed in order to determine the statistical significance of the wetland pollutant removal efficiencies. All p-values calculated were well below 0.05 and the removal efficiencies are therefore considered statistically significant. For this particular ecosystem the findings show that there is no great concern about metal pollution since most of the metals tested for were below the minimum limit for irrigation stipulated by the South African water regulation department (DWAF 1996a). Therefore, the wetland effluent water qualifies for both agriculture and landscape irrigation. Future considerations in choosing to use wetlands as a polishing facility for wastewater treatment systems are highlighted in the study.
The effect of crude water extracts of Tulbaghia violacea Harv. on scaffolds with cardiovascular applications
(Vaal University of Technology, 2020-02) Madike, Lerato Nellvecia; Popat, K. C., Prof.; Pillay, M., Prof.
Tulbaghia violacea Harv. has found extensive uses in traditional medicine for the treatment of numerous ailments among which are tuberculosis, oesophageal cancer, diabetes and cardiovascular diseases. Current reports show that cardiovascular diseases are now the primary cause of mortality worldwide. Thus, the potential of T. violacea plant extracts against cardiovascular diseases should be explored. The objectives of this study were, (i) to conduct qualitative and quantitative preliminary phytochemical screening of T. violacea aqueous leaf extracts, (ii) to conduct Gas chromatography–mass spectrometry (GC-MS) analysis for screening of compounds present in the plant extract, (iii) to evaluate the antioxidant activity of the T. violacea crude extracts using the DPPH:1.1-diphenyl-2-picrylhydrazyl and ABTS: 2,2-azino-bis 3-ethylebenzthiazoline-6-sulfonic acid assays, (iv) to evaluate the antimicrobial activity of the T. violacea crude extracts using disk diffusion and Minimum inhibitory concentration/Minimum bactericidal concentration (MIC/MBC), (v) to evaluate the antithrombogenic properties of T. violacea crude extracts on polystyrene, (vi) to fabricate polycaprolactone (PCL) and PCL-T. violacea incorporated scaffolds, (vii) to evaluate the antithrombogenic properties of T. violacea crude extracts on the fabricated PCL and PCL-T. violacea fabricated scaffolds and, (viii) to evaluate the growth and differentiation of adipose derived stem cells (ADSCs) on the fabricated scaffolds. The qualitative and quantitative phytochemical screening was conducted using standard procedures. Folin-Ciocalteu method was used to evaluate both total phenolic content (TPC) and total tannin content (TTC), the Aluminium chloride method was used for total flavonoid content (TFC) and GC-MS was used to screen for compounds present in the plant extract. The antioxidant activity was evaluated using DPPH and ABTS and the antimicrobial activity was evaluated using disc diffusion and MIC/MBC assays. The antithrombogenic properties of the T. violacea aqueous leaf extracts was then evaluated using platelet activation and whole blood clotting kinetics on polystyrene discs which have been reported to induce platelet activation. The experiment was performed in the absence and presence of 100 and 1000 μg/ml T. violacea plant extracts for both the platelet activation study which used blood plasma and the whole blood clotting kinetics assay which used fresh whole blood. Platelet adhesion was evaluated using fluorescence microscopy and a scanning electron microscope (SEM) was used to evaluate their morphology. Three scaffolds designated as PCL, 10% Tvio and 15% Tvio were fabricated which consisted of a 10% PCL powder and 10% as well as 15% T. violacea aqueous plant extract with respect to the PCL powder weight. The scaffolds were then characterized using Fourier-transform infrared spectroscopy (FTIR) and Energy-dispersive x-ray spectroscopy (EDS). The scaffolds were then evaluated for their antithrombogenic properties in the presence and absence of 100 and 1000 μg/ml T. violacea plant extracts. Platelet adhesion was evaluated using a fluorescent microscope and the morphology was evaluated using SEM. For the cell study, adipose derived stem cells (ADSCs) were cultured on the designed scaffolds and evaluated for their toxicity, viability, adhesion, proliferation, morphology and differentiation into osteoblasts over a period of 3 weeks. Lactate dehydrogenase (LDH) assay was used for toxicity studies, alamar blue assay was used for viability, fluorescence microscopy was used to evaluate cellular adhesion and proliferation while the alkaline phosphate (ALP) assay was used to evaluate differentiation of the cells into osteoblasts. Cell morphology was evaluated using SEM. Phytochemical screening of the prepared T. violacea aqueous extract revealed the presence of terpenoids, flavonoids, cardiac glycosides, saponins, protein, phenols, tannins, carbohydrates and amino acids. This is the first study that has identified the presence of carbohydrates and amino acids in T. violacea aqueous leaf extracts. Different concentrations of 0.1, 1.0 and 10 mg/ml of plant extract were used to conduct the quantitative phytochemical screening assays. There was a concentration dependent increase in the amount of phenols, tannins and flavonoids as the concentration of the plant extracts increased. This was the first study that evaluated the total tannic content of T. violacea plant extracts. The amount of total phenols was higher than that of flavonoids and tannins at every concentration range studied followed by the total flavonoids and lastly total tannins. The GC-MS analysis showed the presence of 33 compounds among which were 2,4 – Dithiapentate - 2,2-dioxide, Cannabidiol, 2,4,5,7 –Tetrathiaoctane and 2,4,5,7 - Tetrathiaoctane 2-dioxide. The presence of sulphur compounds support the characteristic garlic-like smell as well as some of the biological activities of T. violacea plant extracts. The antioxidant activities based on DPPH (0.49 mg/ml) and ABTS (0.24 mg/ml) suggest that T. violacea can be used as potential antioxidant agents. For the antimicrobial activity using disc diffusion, the extracts exhibited appreciable antibacterial activities against Bacillus subtilis, Serratia marcescens, Staphylococcus aureus and S. epidermidis. The highest zone of inhibition was observed for S. epidermidis at 19.50 ± 0.87 mm. The MIC results revealed that the plant extract of T. violacea was moderately active against B. subtilis, S. aureus, S. epidermidis, E. coli, and S. marcescens with MIC value of 2.5 mg/ml. However, the antimicrobial effect of the extract on S. epidermidis was bactericidal when compared to the bacteriostatic effect on the other active microorganisms. The antithrombogenic results on the polystyrene discs showed a significant reduction in the number of platelets that adhered on the polystyrene surfaces treated with plasma mixed with 100 μg/ml of plant extract when compared to the untreated control and the 1000 μg/ml treatment. For the 1000 μg/ml treatment, there was a significant increase in the number of platelets that adhered to polystyrene surfaces. These results were confirmed by the fluorescence and SEM results which showed a higher platelet count for the 1000 μg/ml treatment when compared to the other groups. The whole blood clotting kinetics study showed delayed blood clotting with the 100 μg/ml treatment over a period of 60 min when compared to the untreated control and the 1000 μg/ml treatment. These results correspond with the lower platelet adhesion observation and thus confirm the anticlotting properties of T. violacea aqueous leaf extracts at lower concentrations. The mean diameter of the scaffolds was recorded on the SEM as 275.60 ± 60.65 nm, 193 ± 30 nm and 537 ± 138 nm for the PCL, 10% Tvio and 15% Tvio scaffolds, respectively. The FTIR spectrum revealed the presence of amide groups as well hydroxyl O–H stretching groups which were the characteristic groups for the presence of T. violacea plant extracts in the polycaprolactone. The EDS results showed the presence of potassium, chlorine and sulphur compounds which were only present in the T. violacea scaffolds in addition to the carbon, oxygen and silicon observed in the PCL scaffold. The fabricated scaffolds were then used to evaluate platelet adhesion and activation on blood plasma in the absence and presence of 100 and 1000 μg/ml T. violacea aqueous leaf extracts. The results showed that the 10% Tvio scaffold was more effective in inhibiting platelet adhesion and activation at every treatment group especially when plasma was used in the absence of T. violacea plant extracts. A similar observation to the polystyrene study was observed were addition of 1000 μg/ml of plant extract resulted in the highest number of activated platelets. The study suggests the potential of the 10% Tvio scaffold in the prevention of platelet adhesion and aggregation. The in vitro cell adhesion, proliferation and differentiation of adipose derived stem cells (ADSCs) on the fabricated T. violacea loaded PCL nanofibers was then evaluated. The LDH assay illustrated less activity on the 10% Tvio scaffold when compared to PCL and 15% Tvio scaffolds however, none of the scaffolds were considered as toxic. The alamar blue assay was used for viability after 4 and 7 days of culture. The results showed a significant increase in cell viability for all scaffolds from day 4 to day 7 with the 10% Tvio scaffold having the highest overall cell viability for both day 4 and day 7 of cell cultures. Immunofluorescence staining was then used to count the number of cells using DAPI (4′,6-diamidino-2-phenylindole) stained images and illustrated that the T. violacea incorporated scaffolds supported better cell growth compared to the PCL scaffold. Cell morphology on the T. violacea scaffolds was denser and spread out into cellular extensions when compared to the PCL scaffold after 7 days of cell culture, supporting the higher number of adhered cells from the fluorescence results. For the long term cell study after week 1 and 3, the ALP results showed a significant difference in ALP activity between week 1 and week 3 for all scaffolds. The highest ALP activity was observed for the 15% Tvio scaffolds which is a marker for initial phase of bone matrix deposition. The designed T. violacea scaffolds supported better cell growth compared to the PCL scaffold and their morphology was more spread out and covered the entire surface of the scaffolds after week 3. Lastly, the cell count and osteocalcin differentiation was more prominent on 10% Tvio scaffold indicating higher levels of the protein marker for bone formation. Thus, supporting the use of the 10% Tvio scaffold for long-term cell studies. In conclusion, the results of this study indicated that the aqueous extract of T. violacea is rich is phytochemicals and also possess a broad range of pharmaceutically important compounds which may be attributed to the high antioxidant and antimicrobial activities identified. The results from this study suggest that T. violacea aqueous extracts have antithrombogenic properties at lower concentrations. Scaffolds fabricated with the incorporation of T. violacea plant extract also confirm the potential antiplatelet activity of the fabricated 10% Tvio scaffold. The results also suggest the potential of the fabricated 10% Tvio scaffold to enhance cell adhesion, proliferation and differentiation over long-term cell studies. It can thus be recommended that T. violacea may be useful for tissue engineering applications and bone repair with prospects of preventing cardiovascular diseases associated with bone defects. This research study has provided the foundation for clinical evaluation and outlined the potential effects of T. violacea aqueous leaf extracts as a clinical drug.
To establish the prevalence of MTHFR C677T polymorphism in correlation with homocysteine metabolic markers in a black elderly community, in Sharpeville, Gauteng province in South Africa
(Vaal University of Technology, 2021) Pule, Pule Bongani; Lebea, P. J., Dr.; Grobler, C. J., Dr.
Background: Increased serum homocysteine is well known as an independent cardiovascular risk factor. Hyperhomocysteinemia may be due to several factors such as nutritional deficiencies and genetics. The MTHFR C677T polymorphism is associated with increased serum homocysteine. Folate and vitamin B12 play essential roles in lowering homocysteine levels. Limitations have been identified using serum vitamin B12 as a marker for vitamin B12 status due to lack an efficient of test. Holotranscobalamin has been reported as a more accurate marker for vitamin B12 status. Cardiovascular risk due to hyperhomocysteinemia has been confirmed among the elderly in Sharpeville. Knowledge of the prevalence of MTHFR C677T polymorphism among Black elderlies in South Africa is limited. Objectives: The main aim of the study was to evaluate the prevalence of MTHFR C677T polymorphism as a cardiovascular risk in an elderly black population in Sharpeville. Correlations between the presence of MTHFR C677T polymorphism and homocysteine metabolic markers were evaluated. Holotranscobalamin as a diagnostic test for vitamin B12 status was also assessed in this study. Materials and methods: This study was ethically approved by the Vaal University of Technology ethics committee (20140827-1ms). It was an observational, experimental study conducted in 102 elderly (≥60 years) attending the day-care centre in Sharpeville. Real-Time PCR was used to determine MTHFR genotypes. Folate and vitamin B12 were measured with AIA-PACK. Homocysteine levels were determined with an automated Konelab™ 20i and holotranscobalamin by ELISA. STATA 12 software was used for analysis of descriptive and inferential statistics. Results: The prevalence of MTHFR C677T polymorphism in this sample population was 19%. Heterozygous CT single nucleotide polymorphism was 17% and mutant homozygous TT was 2%. The majority (81%) of the subjects had wild type homozygous CC genotypes. No associations were found between MTHFR C677T genotypes and homocysteine and folate levels. Hyperhomocysteinemia was high (54%) and low (5%) folate deficiency found. No vitamin B12 deficiency was found however 7% were on the category of likely to be deficient. Sensitivity and specificity of holotranscobalamin were 100% and 95% respectively. Conclusion: The conclusions drawn from the study is that the prevalence of MTHFR C677T polymorphism is low within elderly in Sharpeville. There is a high risk of cardiovascular disease as a result of high prevalence of hyperhomocysteinemia. An intervention to lower homocysteine concentration of elderlies residing in Sharpeville is needed. Other genetic predisposing factors of increased homocysteine levels should be investigated.
The effects of solar irradiated Salmonella Typhimurium and campylobacter jejuni on the proliferation and activation of macrophages in vitro
(Vaal University of Technology, 2019-12) Chihomvu, Patience; Ssemakalu, Cornelius Cano, Dr.; Ubomba-Jaswa, Eunice, Dr.; Pillay, Michael, Prof.
Salmonella enterica serovar Typhimurium and Campylobacter jejuni are the leading causes of Salmonellosis and Campylobacteriosis that is characterised by gastroenteritis. These waterborne diseases can be easily prevented by home water treatment methods such as solar disinfection (SODIS). The SODIS process involves placing microbiologically unsafe water in clear plastic or glass bottles and exposing them to direct sunlight for approximately six to eight hours. SODIS kills microbes through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating. The result is microbiologically safe water. Continuous drinking of SODIS treated water may confer some immunological effects on the consumer. These immunological effects have not been thoroughly explored. Therefore, the objectives of this study were to firstly, characterise the effects of solar irradiation on the viability of S. Typhimurium and C. jejuni; secondly, to determine the cytotoxicity and modulation of cell death of solar irradiated S. Typhimurium and C. jejuni on macrophages. Thirdly, to analyse the chemokine and cytokine profiles of macrophages infected with solar irradiated S. Typhimurium and C. jejuni. Lastly, to analyse the host-cell interactions of macrophages infected with solar-irradiated and non-solar irradiated S. Typhimurium and C. jejuni using a proteomic approach. In all the experiments, S. Typhimurium and C. jejuni were (i) heat/chemically treated, (ii) solar and non-solar irradiated for 4 and 8 hours. A murine macrophage cell line RAW264.7 was co-cultured with the differentially treated bacteria species for 3 and 24 hours. Appropriate controls were included. The impact of solar irradiated S. Typhimurium and C. jejuni on intracellular growth, proliferation, cytotoxicity, and apoptosis on macrophages was assessed. Intracellular growth of the both bacterial species was assessed with the gentamicin protection assay, and cytotoxicity was determined by Lactate Dehydrogenase Assay (LDH). The macrophages treated with solar irradiated S. Typhimurium and C. jejuni showed no intracellular growth after 48 hours post-infection. However, the non-irradiated S. Typhimurium survived within the macrophages and were highly toxic to the macrophages (average cytotoxicity of 91%±32). The non-solar irradiated C. jejuni were metabolically active but non-culturable, whereas the solar-irradiated C. jejuni was metabolically inactive. Thus, solar irradiated C. jejuni showed a lower percentage cytotoxicity (2.57% ± 0.32%) in comparison to non-solar irradiated C. jejuni at 24 hours post-infection (p.i.) (30.28% ± 0.05%). Flow cytometric analysis showed that the non-irradiated S. Typhimurium brought about a statistically significant increase in the percentage of necrotic cells (48% ± 2.99%), whereas bacteria irradiated for 8 hours produced a lower percentage of necrotic cells (25% ± 5.87%). The heat/chemical attenuated samples had the lowest percentage of necrotic cells (21.15% ± 5.36%) at 24 h p.i. Macrophages treated with solar irradiated and non-solar irradiated C. jejuni did not induce necrosis, but apoptotic cell death. At 24 h p.i., the highest proportion of apoptotic cell death was observed in macrophages treated with non-solar irradiated C. jejuni whereas the solar irradiated C. jejuni showed a lower percentage of apoptotic cell death. Therefore, there is great possibility that S. Typhimurium and C. jejuni could become avirulent after SODIS treatment and this could prevent gastroenteritis in consumers of SODIS-treated water. The activation of macrophages infected with solar irradiated S. Typhimurium and C. jejuni was also assessed in this study. The production of nitric oxide (NO) was determined using the Greiss Reagent Assay, whereas the production of chemokines, cytokines, and growth stimulating factors by the RAW264.7 cells in vitro was measured using the Luminex 200. The results showed that both solar and non-solar irradiated S. Typhimurium inhibited the production of nitric oxide in the RAW264.7 cells. The heat/chemically attenuated S. Typhimurium induced a significant increase (p<0.0.5) in the production of NO2− in the macrophages when compared to the unstimulated RAW264.7. The chemokine and cytokine levels produced by the macrophages were similar in the solar inactivated S. Typhimurium and the live untreated S. Typhimurium. However, macrophages treated with heat/chemically attenuated S. Typhimurium showed an anti-inflammatory response by inhibiting the production of pro-inflammatory cytokines such as IL-1, IL-1, IL-2, IL-6, and IL-17 in macrophages. The macrophages treated with solar and non-solar irradiated C. jejuni possibly produced an anti-inflammatory effect since the amount of pro-inflammatory cytokines in the samples was significantly reduced during the late infection period (24 h p.i.). This study also analysed the proteomic profiles of macrophages treated with LPS, non-solar irradiated, solar irradiated, heat/ chemical inactivated S. Typhimurium, and C. jejuni. This was carried out using SWATH-mass spectrophotometry-based proteomics. Proteins were extracted from infected macrophages after 24 hours p.i. HILIC-based sample clean-up and digestion, DDA LCMS-MS (spectral library), SWATH LCMS-MS, and data processing were carried out. A total of 15,077 peptides matching to 2,778 proteins were identified at 1% FDR with numerous differentially expressed proteins (DEPs) detected in macrophages treated with lipopolysaccharide (LPS), non-solar irradiated C. jejuni (NS), heat-attenuated C. jejuni (HA) and 4h-solar irradiated (SI4) and 8h-solar irradiated (SI8) C. jejuni, respectively. Pathway analysis revealed that most of the upregulated proteins in macrophages treated with solar irradiated C. jejuni were involved in oxidation-reduction processes, endoplasmic reticulum stress, transport, antigen processing and presentation of exogenous peptide antigens via MHC class I (TAP-dependant) and ATP-biosynthetic processes. The KEGG-pathways also revealed the roles of some upregulated proteins in lysosomal and phagosome pathways. In conclusion, our results revealed that there is coordinated up-regulation of MHC-I processing pathways occurred at 24 h p.i. It is likely that proteins from solar irradiated C. jejuni may undergo proteasomal degradation, and the peptides are transported to the endoplasmic reticulum (ER) and loaded onto MHC-I molecules. Peptide loading results in class I complexes consolidation and transit to the cell surface where antigens can be presented to circulating CD8 + T cells. Additionally, solar irradiated C. jejuni also undergoes degradation in the phagosome. The phagosome has the potential to create antigens that can be expressed on the cell surface of macrophages to stimulate different lymphocytes and induce appropriate immune responses, thus, connecting the innate to adaptive immunity, and this could also have health benefits via the consumption of SODIS treated water. However, proteomic analysis of S. Typhimurium showed no significant differentially expressed proteins in macrophages treated with LPS, non-solar irradiated, and solar irradiated S. Typhimurium. This may be due to an overestimation of the extracted protein. However, DEPs in macrophages treated with heat-attenuated S. Typhimurium showed that macrophages may have adapted an anti-inflammatory M2 phenotype because the IFN-γ signalling pathway was downregulated. This may have contributed to non-expression of the chemokine IFN-γ in RAW264.7 cells. Moreover, proteins such as Hmox1 and Sqstm1 were upregulated, and this is also characteristic of M2 macrophages. This study provided new insights on the effect of solar irradiated Salmonella Typhimurium and Campylobacter jejuni on the proliferation and activation of macrophages in vitro.
Engineering of an enzyme cocktail for biodegradation of petroleum hydrocarbons based on known enzymatic pathways and metagenomic techniques
(Vaal University of Technology, 2020-07) Baburam, Cindy; Tsekoa, T., Dr.; Feto, N. A., Dr.
Hydrocarbon pollution is becoming a growing environmental concern in South Africa and globally. This inadvertently supports the need to identify enzymes for their targeted degradation. The search for novel biocatalysts such as monooxygenases, alcohol dehydrogenases and aldehyde dehydrogenases, have relied on conventional culture-based techniques but this allows sourcing of the biomolecules from only 1-10 % of the microbial population leaving the majority of the biomolecules unaccounted for in 90-99 % of the microbial community. The implementation of a metagenomics approach, a culture-independent technique, ensures that more or less than 100 % of the microbial community is assessed. This increases the chance of finding novel enzymes with superior physico-chemical and catalytic traits. Hydrocarbon polluted soils present a rich environment with an adapted microbial diversity. It was thus extrapolated that it could be a potential source of novel monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH) involved in hydrocarbon degradation pathways. Therefore, the aim of the study was to extract metagenomic DNA from hydrocarbon contaminated soils and construct a metagenomic fosmid library and screen the library for monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH). Accordingly, the fosmid library was constructed from metagenome of hydrocarbon-contaminated soil. Then the library was functionally screened using hexadecane, octadecene and cyclohexane as substrates and fifteen positive clones were selected. The fosmid constructs of the positive clones were sequenced using PacBio next generation sequencing platform. The sequences were de novo assembled and analysed using CLC Genomic Workbench. The open reading frames (ORF) of the contigs were identified by blasting the contigs against uniport database. Accordingly, four novel genes namely amo-vut1, aol-vut3, dhy-sc-vut5 and dhy-g-vut7 that showed close similarity with our target enzymes were further analysed in silico and codon-optimized as per Escherichia coli codon preference. The codon adjusted sequences were synthesised and cloned into pET30a(+) expression vector. However, it is worth noting that expression of amo-vut1 was not successful since it was later identified to be a multi-pass member protein, which made it insoluble despite the use of detergent to the effect. There is a need to meticulously genetically engineer amo-vut1 to remove the signal and other membrane-bound peptides while maintaining its activity. Yet the other three constructs were successfully transformed and expressed in E. coli BL21 (DE3). The enzymes were purified and characterized and cocktail for hydrolysis of hexanol was succesfully engineered based on AOL-VUT3, DHY-SC-VUT5 and DHY-G-VUT7. Therefore, novel enzymes were mined from metagenome of fossil-oil contaminated soil and effective hydrocarbon-degrading enzyme cocktails containing their combination were successfully engineered.
Investigation of seasonal prevalence of low pathogenic avian influenza (LPAI) in a heterogeneous wild waterfowl population in Pretoria
(Vaal University of Technology, 2018-06) Phiri, Thandeka P.; Abolnik, Celia, Prof.; Feto, Naser Aliye, Dr.
Influenza-A virus is a single stranded negative sense RNA virus that is a member of a Orthomyxoviridae group. The virus is diverse and consists of 16 haemagglutinin (H) and 9 neuraminidase (N) glycoproteins subtypes that form a serotype. Avian influenza virus (AIV) has been detected in more than 100 bird species from 26 different families, although Anseriformes and Charadriiformes are considered the natural hosts of the virus. A 12-month study was conducted at the African Pride Irene Country Club lodge in Pretoria where the prevalence of AIV was monitored in a community of wild birds. The African Pride Irene Country Club lodge houses a population of wild bird species such as Egyptian geese (Alopochen aegytptiaca), Yellow-billed duck (Anas undulata), Red knobbed coot (Fulica cristata), African sacred ibis (Threskiornis aethiopicus) and Hadeda ibis (Bostrycha hagedash). A total of 3674 faecal samples were collected over a period of 12 months and screened for AIV group using the Matrix gene (M-gene) real time reverse-transcriptase PCR (rRT-PCR). Positive samples were submitted for virus isolation in embryonated chicken eggs. In addition, the RNAs were screened using H5 and H7 subtype specific rRT-PCR and a conventional universal RCR assay that targets the HA gene was also used. Polymerase Chain Reaction (PCR) products were requenced using Sanger sequencing and the viral isolates were subjected to Next Generation sequencing (NGS). Twenty percent of the samples tested positive for the AIV group and four virus subtypes were identified. One virus isolate was identified through NGS as H3N6; two through conventional PCR and Sanger sequencing as H9Nx and H6Nx. Of the twenty percent samples that tested positive for AIV 98% were identified as H7Nx by subtype specific through rRT-PCR. The highest frequency of AIV positive samples was detected between the months of January and February 2017 (20%), with smaller peaks detected in february and March 2016 (0.3%). Lower peaks were also detected between the months July and November 2016 (0.1%), respectively. A high prevalence of AIV was detected in the late summer months with a frequency of 65% positive, although a low prevalence was also detected in the autumn (0.6%) winter (0.6%) and spring 0.08%). Thus, the study provides a valuable insight into the seasonal prevalence of AIV in a heterogeneous wild duck population in Gauteng Province.
Mining of genes encoding for DNA-manipulating enzymes from hot springs using metagenomic techniques
(Vaal University of Technology, 2019-09) Mokoena, Morena India; Rashamuse, K., Dr.; Feto, Naser Aliye, Dr.
The use of conventional culture-based approach results in vast majority of microbes (90 - 99%) unaccounted for. However, over the past years, the use of metagenomics, which is a culture-independent comprehensive approach has enabled researchers to access nearly 100% of the microbiome. In this study, three hot springs (44 – 70 oC) in Limpopo province of South Africa were investigated as potential sources of genes encoding for DNA-manipulating enzymes (DNA polymerase, DNA ligase and endonuclease), which are central in genetic engineering. They are usually grouped into four broad classes (nucleases, ligases, polymerases and modifying enzymes) depending on the type of the reaction they catalyze. Accordingly, hot spring metagenomic DNA was successfully extracted using modified SDS-CTAB method involving gel purification and electroelution. Consequently, a portion of the extracted metagenomic DNA was used for sequencing and another for fosmid library construction. Sequencing was done using Illumina MiSeq next generation sequencing platform and sequence data analyzed and de novo-assembled using CLC Genomic Workbench, which resulted in 5 681 662 reads and 7 338 contigs. A metagenome expression fosmid library of approximately 2.16 x 103 clones was also constructed using CopyControl™ HTP Fosmid Library Production Kit with pCC2FOS™ Vector. A BLAST algorithm in NCBI revealed 57 distinct genes for DNA polymerase, 29 genes for DNA ligase and more than 100 genes for endonuclease II enzymes. Hence, three genes related to thermophiles representing genes for DNA polymerase, DNA ligase and endonuclease II were selected. Accordingly, the three genes were codon-optimized, synthesized and successfully cloned into pET- 30a (+) and overexpressed in Escherichia coli BL21 (DE3) by inducing with 0.5 mM IPTG and incubating overnight at 16ºC. The cells were lysed using B-PER Reagent, protein extracted and purified using AKTA start protein purification system and purity of 85- 95 % was achieved. From this study, it can be concluded that metagenomics as an approach, can be used to mine for putative DNA-manipulating enzymes from hot spring metagenome. Besides, further study should be conducted to formulate the developed DNA-manipulating enzymes and study the practical application and chart way for commercialization. Moreover, the constructed fosmid library could also be screened for potentially novel thermo-stable biomolecules of industrial and therapeutic importance.
Establishing the correlation between R353Q polymorphism and haemostatic markers in a black elderly community of Sharpeville Gauteng Province South Africa.
(Vaal University of Technology, 2019) Rodrigue, Tagne Wambo Joseph; Lebea, P. J., Dr.; Grobler, C. J., Dr.
Background: In a group of the elderlies (older person) in Sharpeville, Gauteng, South Africa, the majority live in poverty with a poor nutritional status. This makes them susceptible to develop infectious diseases as well as the risk of Chronic Diseases of the Lifestyle (CDL) such as cancer, diabetes, heart attack, obesity and hypertension. One of the most constant features of aging is the progressively elevated levels of coagulation factors such as FVII, fibrinogen, and impairment of fibrinolysis might play a role in the ageing process. These are associated with increased susceptibility to Cardiovascular diseases (CVD) commonly found. An association between elevated levels of FVII and R353Q polymorphism has been established as a risk factor for CVD. This genetic polymorphism R353Q characterizes the substitution in the exon 8 of the FVII gene of guanine-to-adenine, which results in the replacement of arginine (R) by glutamine (Q) in codon 353 of the F7gene. Objectives: The aim of this study was to evaluate the prevalence of R353Q polymorphism in correlation with haemostatic markers within an urban elderly community in South Africa. Method: This study was ethically approved, and it is an experimental research design on the prevalence of R353Q polymorphism in correlation with homeostatic status (Factor VII, Fibrinogen and PAI-1). The study was done in a black elderly population living in the Vaal triangle region of Sharpeville, attending a day care center, who gave consent to participate in the study. A purposely selected sample of 102 subjects, who met the inclusion criteria were used. The homeostatic status was measured by factor VII and fibrinogen measuring coagulation and PAI-1 measuring fibrinolysis. Results: The prevalence of R353Q genetic polymorphism was established in 14.5% of the sampled population. The prevalence of the RQ (AG) genotype was determined in the sample population with 6.5 % of elevated factor VII levels, 7.8% of increased fibrinogen levels (coagulation) and 10.5 % of decreased levels of PAI-1. The R(A) allele, was detected in 1.3% of the sampled population of normal levels of FVII, fibrinogen and PAI-1. The dominant allele G(Q) was present in 76.3% of the sampled population. An imbalance haemostatic marker was established in the sampled population with 61% of elevated levels of factor VII, 70% of elevated levels of fibrinogen and 88% had a decreased level of PAI-1. Conclusion: The prevalence of R353Q polymorphism was established in this sample population, having an imbalanced haemostatic status of hypercoagulation (factor VII and fibrinogen) and imbalance fibrinolysis (PAI-1), which are strongly associated to CVDs.
Sequence and function based screening of the goat rumen metagenome for novel amylases
(Vaal University of Technology, 2019-09) Rabapane, Kgodiso Judith; Nelson, K. E., Prof.; Feto, Naser Aliye, Dr.
During one of our preliminary studies in 2015, metagenomic DNA extracted from the goat rumen was sequenced and the in silico mining of the biorefining enzyme showed the presence of significant number of different biocatalysts, such as amylases (E.C 3.2.1.1), xylanases (E.C 3.2.1.8), pectinases (E.C 3.2.1.15) and cellulases (E.C 3.2.1.4). Hence, a subsequent study was conducted which is aimed at extracting metagenomic DNA from the goat rumen, constructing the metagenomic library using pCC2-FOS™ plasmid vector (Epicentre®), and eventually screening the constructed library for potential novel amylases using soluble starch as a substrate. Accordingly, rumen digesta was aseptically collected from four compartments of each goat and pulled before extraction of metagenomic DNA. The conventional CTAB protocol was modified to extract the metagenomic DNA from the rumen digesta. As a result, high molecular weight DNA was obtained and used to construct the metagenomic fosmid library. Since the host (Escherichia coli EPI 300-T1r) supplied with CopyControlHTP Fosmid Library Production Kit has background amylase expression we opted for a knockout E. coli strain with deleted starch hydrolysis (amylase expression) pathway. The library was subsequently screened for the presence of amylase isoforms using soluble starch as a substrate. Therefore, for the purpose of this study, four fosmids clones showing amylase activity were selected, recombinant vector isolated and MiSeq-sequenced. Out of four recombinant proteins, only one (pET30a(+)-amy-vut12) was successfully expressed. Subsequently, pET30a(+)-amyvut12 was further characterize physicochemically. Interestingly, the recombinant enzyme showed maximum activity in the pH and temperature ranges of 6.0 - 8.0 and 70 - 90oC, respectively. Hence, this implies that novel recombinant protein has sound activity from acidic to alkaline pH range and potently thermostable. Further work should be done to optimize and improve the solubility of three other recombinant proteins (pET30a(+)-amy-vut2, pET30a(+)-amy-vut9 and pET30a(+)-amy-vut14) studied, which might harbour important traits. Most importantly, immobilization as well as crystallographic studies of pET30a(+)-amy-vut12 and downstream applications should further be investigated.
The evaluation of whole blood cytokine assay for diagnosis of M.tuberculosis infection in South African children with household tuberculosis contact
(Vaal University of Technology, 2019-04) Masilo, J. M.; Adrian, P. V., Dr.; Grobler, C. J., Dr.
Background: There are critical unmet needs for improved strategies in the detection and diagnosis of M.tuberculosis infection in children, and for prevention of tuberculosis disease in children. Bacillus Calmette-Guérin (BCG) vaccination has limited the utility of tuberculin skin testing (TST) in areas with high vaccine coverage. Objectives: The aim of this study was to estimate the prevalence of M.tuberculosis infection in children with household tuberculosis contacts, using QFT-GIT testing in comparison with TST. Methods: This study was a cross-sectional design to assess the performance of a new T-cell based blood test, namely QuantiFERON-TB Gold In Tube (QFT-GIT), for diagnosis of tuberculosis infection in the children (n=182) of adults (n=124) with pulmonary tuberculosis, additionally to determine the prevalence of M.tuberculosis infection in children with household tuberculosis contacts, using QFT-GIT testing in comparison with TST. The study was carried out at Chris Hani Hospital. For children involved in the study, tuberculosis exposure information was obtained, together with TST, QFT-GIT, and HIV testing. Data obtained from both experiments was statistically analysed using SPSS version 24 to determine whether there was a significant agreement between QFT-GIT and TST on the detection of M.tuberculosis prevalence in children with house hold contacts with confirmed M.tuberculosis infection. Results: This study examined the sensitivity and specificity of the QFT-GIT tests compared with the standard TST for diagnosing latent tuberculosis disease in paediatric contacts. Because of the lack of a latent tuberculosis "gold standard”, the specificity and sensitivity of QFT-GIT was calculated with a two-by-two table method. The specificity of the QFT-GIT was 84% and the sensitivity was 85%. There was a good correlation between QFT-GIT and TST (Cohen’s kappa of 0.705). Seventeen percent (17%) of the 182 children tested by QFT-GIT yielded indeterminate results. Age was associated with indeterminate QFT-GIT results in paediatric tuberculosis contacts. Point prevalence for QFT-GIT was recorded as 31% at baseline and 39.5% after six months indicating variability between QFT-GIT results at baseline and after six months. Conclusion: It was concluded that the prevalence of tuberculosis infection was common among South African children who live with an adult with active tuberculosis. The agreement between QFT-GIT assay and TST for the diagnosis of latent tuberculosis in children was high. Although TST and QFT-GIT assays appeared comparable, QFT-GIT showed higher positivity rate amongst those contacts with reported household tuberculosis exposure compared to TST. The QFTGIT assay was a better indicator of the risk of M.tuberculosis infection than TST in a BCG-vaccinated population.
Correlating the prevalence of C174G polymorphism with IL-6, TNF-α and Hs-CRP in an elderly black South African population
(Vaal University of Technology, 2019-03) Valentine, Jessica; Lebea, J., Dr.; Grobler, C. J., Dr.
Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and the prevalence thereof is on the rise in developing countries due to the demographic transition and urbanization. The inflammatory process, atherosclerosis, is at the root of the majority of CVDs and is caused by unresolved inflammation. Various cardiovascular risk factors such as hyperglycaemia, dyslipidaemia, hypertension, smoking and aging stimulate the development of atherosclerosis through triggering inflammation. Being in a state of chronic low-grade inflammation therefor places an individual at higher risk of developing CVD, with inflammation playing a cause and effect role. The aim of this study was to investigate the inflammatory status of an elderly black South African population by analysis of inflammatory markers HS-CRP, TNF-α and IL-6, as well as the genetic polymorphism C174G associated with increased serum levels of IL-6 in some populations. The research was conducted in the field of Biomedical Sciences as a quantitative, cross-sectional, analytical observational design. The study was ethically approved and involved collection of 84 blood samples from volunteers in a purposively selected population as part of a larger collaborative study. Serum was used to analyse HS-CRP, TNF-α and IL-6 and DNA was extracted from whole blood for analysis of the C174G polymorphism. The median serum HS-CRP of 6.44mg/L (IQR = 2.82 - 9.86mg/L) fell within the highest risk (>5mg/L) of CVD and 75% of participants were at high (3.01-5mg/L) or very high (>5mg/L) risk. The median TNF-α of 0.00pg/mL was within the normal range and only 2.6% of participants had high serum TNF-α levels. The median serum IL-6 level was 1.92pg/mL and was also within the normal range with only 2.6% of participants who had high serum IL-6 levels. For the C174G polymorphism analysis, 98.6% had the GG, 1.4% the GC genotype and no participants had the CC genotype. The median serum IL-6 level of the homozygous GG group was 6.51mg/L, higher than the 4.13mg/L serum IL-6 of the heterozygous GC group. The difference in IL-6 should be considered with caution as only one participant had the C allele. A highly significant (p=0.001) correlation was found between HS-CRP and IL-6, as well as between IL-6 and TNF-α (p = 0.048). The elderly black Sharpeville community is in an increased inflammatory state which puts them at risk of CVD. The prevalence of the C allele in the C174G polymorphism is low in this population. Further research could be conducted as intervention studies to decrease the inflammatory state of the population and influence health policy changes to improve prevention of CVD.

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