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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.
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 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 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.
Assessing the genetic diversity of Alternaria Bataticola in South Africa using molecular markers
(Vaal University of Technology, 2015) Chalwe, Joseph Musonda; Adebola, Patrick, Dr.; Pillay, Michael, Prof.
Sweetpotato (Ipomoea batatas) is an important food crop that is grown in many countries. A number of viral and fungal sweetpotato diseases have been reported worldwide. One of the major and most economic diseases of the sweetpotato is Alternaria blight which is caused by the fungal pathogen Alternaria bataticola. This disease can be managed in a short term using fungicides and cultural practices. However, a long term and inexpensive approach is through the development of resistant cultivars. A prerequisite to this approach is the knowledge of the genetic diversity of this fungal pathogen. This study assessed the genetic diversity of 25 South African isolates of A. bataticola from naturally infected leaves and stems collected from different sweetpotato growing regions in South Africa by (i) characterising the isolates based on their morphology (ii) pathogenicity tests (iii) random amplified polymorphic DNA (RAPD) (iv) variation of the ITS2 sequences and (v) prediction of the ITS2 secondary structures. The isolates revealed some variation in colony colour pigments after culturing but Koch’s postulates were confirmed by their pathogenicity tests. The analysis of RAPD and variation of the ITS2 sequences showed high levels of variation (100%) among the isolates. Dendrograms generated from these analyses had many subclusters and did not cluster the isolates according to their geographic origins. The ITS2 secondary structures were predicted and can be used to identify and distinguish the isolates. This information in addition to the genetic diversity of the A. bataticola isolates will aid plant breeders in the development of resistant sweetpotato cultivars and early management of blight disease in South Africa.
Assessing the genetic diversity of South African sweetpotato germplasm using DNA and protein markers
(Vaal University of Technology, 2013-06) Selaocoe, Maleshoane Ellen; Adebola, Patrick, Dr.; Pillay, Michael, Prof.
Sweetpotato is one of the most important food crops in developing countries including South Africa. Currently two major types of cultivars are grown in South Africa: one is the orange-fleshed sweetpotato (OFSP) which has high β-carotene content, a precursor of vitamin A. The second type is the cream-fleshed sweetpotato (CFSP) which has low β-carotene content but is high in dry matter. Most South Africans prefer the CFSP although the OFSP offers more advantages. This presents a challenge to plant breeders to develop new varieties that will combine the desirable qualities of both the cultivars. To achieve this goal, plant breeders need knowledge about the genetic variation of the crop to develop an efficient breeding programme. This study assessed the genetic relationships of 28 orange- and cream-fleshed sweetpotato accessions by (i) examining the variation in leaf proteins, (ii) using random amplified polymorphic DNA (RAPD) and, (iii) using variation of the ITS region. The analysis of proteins, RAPD and variation of the ITS region polymorphism levels were 55.6%, 98% and 16.5%, respectively. Dendrograms generated from all the analyses generally clustered the accession according to their flesh colour and country of origin. Analysis of molecular variance (AMOVA) found a significant difference between OFSP and CFSP and a significant difference between the South African and non-South African germplasm. The high genetic diversity in the South African sweetpotato germplasm is a positive indicator for a breeding programme that has a number of targets such as breeding for nutritional improvement, disease resistance and drought tolerance
Assessing the morphological variation and characterising the proteins of bambara groundnut (Vigna Subterranea L. Verdc)
(Vaal University of Technology, 2016-12) Evangeline, Unigwe Amara; Adebola, P., Dr.; Pillay, M., Prof.
Bambara groundnut (Vigna subterranea L. Verdc) is an underutilized crop in the African continent. It is a drought tolerant crop and fixes atmospheric nitrogen. Bambara groundnut is primarily grown for the protein content of its seeds and is mainly produced by small scale farmers at the subsistence level. However, despite its importance as a subsistence crop in many African countries, only local landraces of bambara groundnut are still cultivated. Mass selection of a few local varieties for the main agronomic characteristics has been carried out. All the bambara groundnut germplasm in South Africa has not been morphologically characterized. Although the protein of bambara groundnut is of good quality and is rich in lysine, there is no information on the characterisation of these proteins. The presence of antinutritional factors in the crop has also received little attention. This study focused on three major objectives including: (I) to assess the extent of morphological variations among thirty selected landraces of bambara groundnut, (II) to characterize the major seed proteins in these accessions using one dimensional gel electrophoresis, and (III) to determine the presence of any anti-nutritional factors in the seeds of the selected bambara groundnut landraces. 30 accessions of bambara groundnut were evaluated for their variability in agronomic and morphological traits. The field experiment was conducted at ARC-VOPI in Roodeplaat research farm during the 2014/2015 summer cropping season. The field trial was arranged as a complete randomized block design with 3 replications. 18 quantitative traits were recorded to estimate the level of genetic variability among accessions. 4 different methods were employed to extract seed proteins from 30 bambara groundnut accessions in order to ascertain the best method for protein extraction. These methods included: 10%-80% isopropanol, 10% trichloroacetic acid (TCA) in acetone solution, sonication and 2x Lammeli buffer extraction methods. The quick start Qubit® fluorometer protein kit was used to determine the protein concentration in each sample. The samples were then subjected to one dimensional gel electrophoresis. For antinutritional analysis, 5 factors (condensed tannins, free and phytic acid phosphate, polyphenol and trypsin contents) were used to determine the amount of antinutrient in 30 bambara seeds that were ground to a fine powdery flour. 3 replicates of all the samples were ground for each assay evaluated. The flour was then immediately extracted and used for the different assays. The analysis of variance revealed significant differences only in 10 of the 18 phenotypic traits that were evaluated. The UPGMA cluster analysis based on the quantitative traits produced four distinct groups of genotypes and a singleton. Genotypes SB11-1A, SB19-1A, SB12-3B and Bambara-12 were found to possess good vegetative characters and are recommended for use as suitable parents when breeding cultivars for fodder production. Desirable yield and yield-related traits were identified in B7-1, SB4-4C, SB19-1A, Bambara-12 and SB16-5A and are recommended as suitable parental lines for bambara groundnut grain production improvement. The quantitative characters therefore provided a useful measure of genetic variability among bambara genotypes and will enable the identification of potential parental materials for future breeding programmes in South Africa. Out of the 4 different seed protein extraction methods exploited for this study, the 2x Laemmli buffer extraction method produced the best result with clear protein bands. A unique feature from all extraction methods was the presence of a common protein band at ̴ 75 kDa. All extraction methods except 10 % TCA-Acetone resolved common banding patterns in all the bambara groundnut samples. This data suggests that there is very little or no intraspecific genetic diversity among the seed proteins of bambara groundnut accessions studied. There was wide variation in the content of the five antinutritional compounds among the thirty bambara groundnut accessions. The mean values for condensed tannin content ranged between 0.20 - 6.20 mg/g. Free phosphate recorded an overall mean of 1.71 mg/g while a range of 1.35 - 4.93 mg/g was observed by phytic acid phosphate (PAP). The polyphenol content had an overall mean of 0.39 mg/g and trypsin inhibitor (TIA) was quite variable among the bambara groundnut accessions ranging from 5.30 - 73.40 TIA/mg. Generally, higher levels of antinutrients were observed in this study compared to the other studies. The results obtained in this study led to a conclusion that although variations exits among the accessions studied, further research is required to verify the extent of morphological variations, the efficiency of protein extractions methods evaluated and the effects of these antinutrients in human and animal feeds.
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.
Biochemical and molecular characterization of heavy metal resistant bacteria isolated from the Klip River, South Africa
(Vaal University of Technology, 2014) Chihomvu, Patience; Stegmann, P., Dr.; Pillay, M., Prof.
The Klip River has suffered severe anthropogenic effects from industrial, agricultural, mining and domestic activities. As a result harmful contaminants such as heavy metals have accumulated in the river, causing microorganisms inhabiting the environment to develop mechanisms to protect them from the harmful effects of the contaminants. The current study deals with the isolation and characterization of heavy metal resistant bacteria isolated from the Klip River Catchment. Water and sediment samples were collected from 6 sites of the Klip River, and the Vaal Barrage (control). In-situ parameters, such as pH, turbidity, salinity, conductivity, temperature and dissolved oxygen were determined. Lead, iron, cadmium, nickel, zinc and copper concentrations of water were determined by atomic absorption spectroscopy. For bacterial analysis sediment and water samples were collected in sterile glass jars and bottles respectively. Heavy metal resistant bacterial isolates were screened on heavy metal constituted Luria Bertani (LB) agar. Biochemical profiles of the isolates were constructed using the API 20E® strips, antibiotic susceptibility tests were done and growth studies were carried out using spectrophotometric methods. The isolates were identified using 16SrDNA sequencing and alignment. A partial sequence of the copper resistance gene pcoA was amplified from strains Lysinibacillus sp. KR25 [KJ935917], and Escherichia coli KR29 [KJ935918]. The pcoR gene was amplified from E. coli (KR29) and the partial sequence for the chromate resistance gene chrB, was amplified from Pseudomonas sp. KR23 [KJ935916]. The gene fragments were then sequenced and translated into protein sequences. The partial protein sequences were aligned with existing copper and chromate resistance proteins in the Genbank and phylogenetic analysis was carried out. The physico-chemical properties of the translated proteins were predicted using the bioinformatics tool Expasy ProtParam Program. A homology modelling method was used for the prediction of secondary structures using SOPMA software, 3D-protein modelling was carried out using I-TASSER. Validation of the 3D structures produced was performed using Ramachandran plot analysis using MolProbity, C-score and TM-scores. Plasmid isolation was also carried out for both the wild type strains and cured derivatives and their plasmid profiles were analysed using gel electrophoresis to ascertain the presence of plasmids in the isolates. The cured derivatives were also plated on heavy metal constituted media. Antibiotic disc diffusion tests were also carried out to ascertain whether the antibiotic resistance determinants were present on the plasmid or the chromosome. The uppermost part of the Klip River had the lowest pH and thus the highest levels of heavy metal concentrations were recorded in the water samples. Turbidity, salinity and specific conductivity increased measurably at Site 4 (Henley on Klip Weir). Sixteen isolates exhibiting high iron and lead resistance (4 mM) were selected for further studies. Antibiotic susceptibility tests revealed that the isolates exhibited multi-tolerances to drugs such as Ampicillin (10 μg/ml), Amoxcyllin (10 μg/ml), Cephalothin acid (30 μg/ml), Cotrimoxazole (25 μg/ml), Neomycin (30 μg/ml), Streptomycin (10 μg/ml), Tetracycline (30 μg/ml), Tobramycin (10 μg/ml) and Vancomycin (30 μg/ml). Growth studies illustrated the effect of heavy metals on the isolates growth patterns. Cadmium and chromium inhibited the growth of most of the microorganisms. The following strains had high mean specific growth rates; KR01, KR17, and KR25, therefore these isolates have great potential for bioremediative applications. Using 16SrDNA sequencing the isolates were identified as KR01 (Aeromonas hydrophila), KR02 (Bacillus sp.), KR04 (Bacillus megaterium), KR06 (Bacillus subtilis), KR07 (Pseudomonas sp), KR17 (Proteus penneri), KR18 (Shewanella), KR19 (Aeromonas sp.), KR22 (Proteus sp.), KR23 (Pseudomonas sp.), KR25 (Lysinibacillus sp.), KR29 (Escherichia coli), KR44 (Bacillus licheniformis) and KR48 (Arthrobacter sp.). Three heavy metal resistance genes were detected from three isolates. The pcoA gene was amplified from strains Lysinibacillus sp KR25, and Escherichia coli KR29; pcoR gene from E. coli KR29 and the chrB gene, from Pseudomonas sp. KR23. The genes encoding for heavy metal resistance and antibiotic resistance were found to be located on the chromosome for both Pseudomonas sp. (KR23) and E.coli (KR29). For Lysinibacillus (KR25) the heavy metal resistance determinants are suspected to be located on a mobile genetic element which was not detected using gel electrophoresis. The translated protein sequence for pcoA_25 showed 82% homology with the copper resistant protein form Cronobacter turicensis [YP003212800.1]. Sequence comparisons between the pcoR partial protein sequence found in E. coli KR29 showed 100% homology with 36 amino acids (which was 20% of the query cover) from a transcriptional regulatory protein pcoR found in E. coli [WP014641166.1]. For the chrB partial protein sequence detected in Pseudomonas sp. (KR23), 97% of the query sequence showed 99% homology to a vitamin B12 transporter btuB in Stenotrophus sp. RIT309.
Characterisation of Amaranthus Tricolor mutant plants with increased drought-tolerance
(Vaal University of Technology, 2010-02) Kgang, Itumeleng Eugenia; Van Emmenis, Lynelle, Dr.; Laloo, Neelan
Amaranthus tricolor (A. tricolor) is a nutritious vegetable crop that is used as a subsistence and cash crop in the rural areas in Africa. Its yield and production is severely limited by abiotic stresses such as drought. Mutation technology, using gamma irradiation, was previously employed as a tool to create genetic variation in order to select for lines with improved drought-tolerance. During irradiation, 160 Gy (Gray) was selected as the optimal dosimetry that allowed subsequent seed germination. The resulting mutant lines were screened over several generations under field and greenhouse conditions and seven promising drought-tolerant lines were selected. Here we report on physiological and morphological studies of two of these Amaranthus mutant lines (#2 and #5) to confirm the enganced drought-tolerance. Plants were grown in the greenhouse in plastic pots containing germination mix with fertiliser. They were exposed to 21 days of well-watered condition, 19 days of drought-stress conditions and 7 days of re-watering. shoot height, leaf area, protein content and relative water content (RWC) of the fresh and dry material were determined colorimetrically under well-watered and drought-stress conditions, while anthocyanin was only measured during well-watered conditions. Shoot height, leaf area, number of leaves per plant and the protein content were significantly reduced under water-stress conditions. Under well-watered condition mutant #5 grew faster with the shoot length significantly higher than mutant #2 and the wild type. Even though drought adversely affected shoot lenght, mutant#5 still performed better than mutant #2 and the wild type under drought-stress conditions. While under both well-watered and drought-stress conditions, the wild type plants had bigger leaf area compared to the two mutant lines. After 16 days of drought-stress conditions, all the leaves of the wild type plants were dried out, as a result no wild type plants recovered after 8 days re-watering. Meanwhile, both mutant #2 and #5 plants recovered significantly after 8 days of re-watering. The wild type was tolerant compared to the two mutant lines. Protein content for mutant #2 plants was higher under both well-watered and drought-stress conditions but was not significantly different from mutant #5 plants compared to the wild type plants after 19 days of drought-stress conditions. Furthermore, genetic diversity was examined in all the Amaranthus lines using random amplified polymorphic DNA (RAPD) analysis. Nineteen arbitrary RAPD markers were used of which two detected polymorphisms (OPA) 07 and OPA 16).
Characterization and identification of microbial communities in pigeon droppings using Culture-Independent techniques
(Vaal University of Technology, 2015-08) Leareng, Samuel Keeng; Feto, Naser Aliye, Dr.; Pillay, M., Prof.
Pigeon droppings, found in abundance in most cities and towns where pigeons are found, are a source of potential yeast and molds into the environment. Invasive fungal infections are a cause of morbidity and often mortality in immunocompromised individuals. The objective of this study was to the identification of bacterial and mold agents from pigeon droppings. Pigeon droppings samples were collected from three locations during the winter and summer months and studied for the occurrence of bacteria, yeast and molds by utilising culture-independent techniques. Amplification of the 16S rDNA gene and the internal transcribed spacer (ITS) region, cloning and ARDRA and DGGE were used for the characterisation of the microbial populations followed by sequencing. Several mold and yeasts, as well as bacteria were found to be present in pigeon droppings, which can spread into the environment and be transmitted to immunocompromised individuals and children. DGGE analysis of the bacterial communities revealed banding patterns that clustered all but one winter samples and all summer samples, showing a high similarity among the microbial members in both seasons and sample locations. Fungal DGGE analysis revealed clusters that grouped summer and winter samples from Johannesburg and Pretoria while VUT samples were clustered on their own. From the identification of fungal and bacterial DNA, Cryptococcus species was the majority of fungi isolated from the dropping samples. Geotrichum, Kazachstania and Fusarium species were isolated from phylotypes obtained from ITS amplicons analysed by ARDRA. Lactobacillus and Enteroccoccus species, organisms usually found in the gastrointestinal tract were the common bacterial members identified. The results showed no difference in microbial communities across all sample locations, while seasonal changes also had no impact in microbial community patterns.
Co-production of inulinase by Kluyveromyces marxianus and Saccharomyces cerevisiae in solid state fermentation
(Vaal University of Technology, 2014-02) Molefe, Nnana Mantsopa; Reddy, P.; Padayachee, T., Prof.
Solid-state fermentation (SFF) has emerged as a good method for the production of microbial enzymes such as inulinases. The use of low-cost agricultural plants and agro-industrial residues as substrates in SSF processes provides a value adding alternative to these otherwise under/or un-utilised vegetation. Production of inulinases, using various inulin-containing plant materials as carbon sources was studied using pure and mixed cultures of yeast strains. All substrates resulted in different levels of enzyme activity. A mixed culture of Kluyveromyces marxianus and Saccharomyces cerevisiae produced an extracellular exoinulinase when grown on different types of inulin-containing plant materials. Initial inulinase production was achieved as follows: 10 IU/gds (garlic cloves), 15 IU/gds (parsnips), 10 IU/gds (wheat bran) and 7 IU/gds (amadumbe) by K. marxianus and S. cerevisiae in a mixed culture. The production of inulinases by a mixed culture of K. marxianus and S. cerevisiae under SSF was further optimized by investigating initial moisture content, temperature, carbon source, nitrogen source, inoculum volume and inoculum ratio. The highest inulinase activity attained was in garlic cloves (85 IU/gds), followed by parsnips (65 IU/gds), wheat bran (37 IU/gds) and amadumbe (25 U/gds). The activities yielded 5.6 fold higher inulinase than in preliminary studies. The optimum pH and temperature of the crude enzyme were 5.0 and 50 oC, respectively. The pH and temperature stability of the enzyme was steady for 1 hour retaining about 64% activity. The average inulinase/invertase activity (I/S) ratio of 1.0 by crude inulinases was also observed after 48 hours. The crude extracellular enzyme extracts from the garlic cloves, parsnips, amadumbe and wheat bran were partially purified by ammonium sulphate precipitation and showed a specific activity of 9.03 U/mg, 0.08 U/mg, 4.12 U/mg and 0.133 U/mg respectively. The Km and Vmax values of the inulinase were 21.95 mM and 2.09 μM/min; 19.79 mM and 1.38 μM/min; 31.59 mM and 0.51 μM/min; and 25.74 mM and 0.23 μM/min, respectively. All extracts demonstrated potential for large-.scale production of inulinase and fructose syrup.
A Comparative study between the prevalence of MTHFR A1298C SNP and homocysteine metabolism in an elderly black South African population
(Vaal University of Technology, 2018-08) Dippenaar, Luzanne; Lebea, P. J., Dr.; Grobler, C. J., Dr.
Background: Cardiovascular diseases are one of the most common causes of death worldwide. This is not only a problem in developed countries, it is of major concern for public health in developing countries as well. Increased homocysteine is an independent risk factor for cardiovascular diseases. Nutritional deficiencies of folate, vitamin B6 and vitamin B12 are associated with hyperhomocysteinemia. MTHFR A1298C, a single nucleotide polymorphism, is similarly linked with higher concentrations of homocysteine. The aim of this study was to determine the prevalence of MTHFR A1298C in a black elderly population, along with folate, vitamin B6 and vitamin B12 and to evaluate the effect on homocysteine levels. Methodology: The research design was an observational cross-sectional study and was ethically approved. A total of 84 elderly who attend a day-care centre (also met inclusion criteria) were purposively selected. DNA was extracted and frozen on the day of blood collection. The MTHFR A1298C genotype was determined with real time PCR. Homocysteine, folate, vitamin B6 and vitamin B12 serum levels were detected with commercial assay kits. Results: Homocysteine was found to be elevated with a median of 17.78 µmol/L (interquartile range 13.98-21.03 µmol/L). Serum folate, vitamin B6 and vitamin B12 medians were in the normal range. Although, 5.95% and 22.62% of the population were deficient and possibly deficient for vitamin B12, respectively. MTHFR A1298C frequency was as follow: 89.29% (AA), 9.52% (AC) and 1.19% (CC), with no significant correlation (p>0.05) with homocysteine. Vitamin B12 correlated significantly with homocysteine levels. Conclusion: Vitamin B12 deficiency had an effect on homocysteine levels. Overall, nutritional deficiencies are not responsible for the hyperhomocysteinemia in this population. In conclusion from this study showed MTHFR A1298C frequency in black South Africans does not contribute to homocysteine as a risk factor for cardiovascular disease. Keywords: Cardiovascular disease, elderly, folate, homocysteine, MTHFR A1298C, vitamin B6, vitamin B12.
Comparative study of the immunomodulatory effect of solar and photonically inactivated salmonella enteritidis on dendritic cells in-vitro
(Vaal University of Technology, 2022) Adeniran, Dorcas Oluwaseun Taiwo; King, Abia Akebe Luther, Dr.; Ssemakalu, Cornelius Cano, Dr.
Salmonellosis is a food and water-borne disease that affects humans, especially those that are immunocompromised as well as children and the elderly. This disease is caused by a variety of Salmonella species. Salmonella Enteritidis (SE) is the most frequently isolated serovar in infections occurring in humans and from animals all over the world. Salmonella Enteritidis is found in many animals and can survive in environmental samples for several weeks under ideal conditions. The failure of waste water treatment plants, agricultural pollution, and storm water runoff into natural water sources has led to an increase in the presence of Salmonella in water. The possibility of fecal contamination of water remains high in resource poor communities where sanitary and hygienic practices are inefficient or insufficient. However, many resource poor communities are using solar disinfection (SODIS) as a means of treating water prior to consumption. The SODIS method is achieved by exposing bacterial contaminated water to the sun for the period of 6 to 8 hours. The reliability of the SODIS process depends on factors such as temperature, dissolved oxygen and most importantly UV-A radiation. These factors cannot be controlled in a natural environment due to fluctuations or climatic changes in weather conditions. Instead of relying only on SODIS, other methods such as the use of a photonic device to disinfect microbiologically water are being used. The main aim of this study is to compare the immunomodulatory effect of solar irradiated and photonically inactivated S. Enteritidis on dendritic cells in-vitro and to provide supporting information on the immunological benefits on the consumers of SODIS drinking water through a SODIS mimicking device. To achieve this aim, there was a need to optimize the SODIS and photonic inactivation conditions of S. Enteritidis. Salmonella Enteritidis cultures were exposed to solar irradiation during spring, summer and winter as well as photonically using an ultraviolet light. The result revealed that the inactivation efficiency of Solar ultraviolet radiation (SUVR) on S. Enteritidis was season dependent. A total loss of activity was observed in S. Enteritidis during summer and no regrowth was observed. With the photonic device, a combination of UV and oxygen inactivated the S. Enteritidis to below detectable limits. This study compared the protein profiles of solar irradiated and photonically inactivated S. Enteritidis using SDS-PAGE. The results showed a gradual decrease in the concentration of the protein banding patterns with time in S. Enteritidis that was either solar irradiated or photonically inactivated. The ability of the solar and photonically inactivated S. Enteritidis to induce maturation of dendritic cells in-vitro was also investigated. There was a significant increase in CD80 when the 8-hour solar inactivated samples of S. Enteritidis was used to stimulate the dendritic cells. The higher levels of co-stimulatory molecules observed suggested the possible involvement of these molecules in antigen uptake and presentation to produce a specific immune response. This finding will contribute towards the understanding of the immunological effects that may be generated from consuming SODIS water and whether it may result in an immune reaction or response. Although the current study shows that solar irradiated and photonically inactivated cultures of S. Enteritidis were able to induce the expression of key immunological surface makers by dendritic cells, further studies are required to corroborate the findings of this study.
The construction and evaluation of a novel tubular photobioreactor at a small pilot plant scale
(Vaal University of Technology, 2012-07) Kutama, Makonde; Van Wyk, C.; Stegmann, P., Dr.
The mass production of algae for commercial purposes has predominately been carried out in open ponds systems. However, open ponds systems have a number of disadvantages such as poor light utilization, requirement for large areas of land and high risks of contamination. On the other hand, photobioreactors have attracted much interest because they allow a better control of the cultivation conditions than open systems. With photobioreactors, higher biomass productivities are obtained and contamination can be easily prevented. Photobioreactors can also be engineered to manipulate the light and dark photosynthetic reactions thus enhancing biomass productivity. The main objective of this study was to construct a novel tubular photobioreactor which had the ability to expose the cultured alga to light and dark phases with the aim of optimizing the algal biomass production. A novel tubular photobioreactor with the ability to manipulate the cultured alga’s light and dark photosynthetic reactions was constructed in this study. The alga Spirulina platensis was chosen as the test organism in this novel tubular photobioreactor due to a number of reasons such as its globally socioeconomic importance, its tolerance of higher pH and temperature values which makes it almost impossible to contaminate. The cultivation process of Spirulina in the photobioreactor was investigated through alternating light and dark cycles in an attempt to increase the photosynthetic efficiency of the culture. The effect of different light intensities on the growth of Spirulina in the novel tubular photobioreactor was investigated and it was found that the best light condition that favored higher biomass formation was at 600 μ mol m-2 s-1. Five different light/ dark ratios were evaluated at a light intensity of 600 μ mol m-2 s-1 during a batch mode of operation of the novel tubular photobioreactor. The light/ dark ratio of 1:0.25 was found to be the best ratio because it gave the highest biomass in the shortest period of time when compared to the other ratios used. These results seem to suggest that longer light cycle relative to dark cycle results in higher biomass production. The ratio of 1:0.25 was then used to operate the novel tubular photobioreactor in a continuous mode. A maximum biomass productivity of 25 g/m2/day was achieved which corresponded to a net photosynthetic efficiency of 5.7 %. This result was found to be higher than what most photobioreactors could achieve but it was 2.8 g/m2/day lower than the highest ever reported productivity in a photobioreactor when Spirulina is cultivated. The 2.8 g/m2/day lower was attributed to the different materials used in the construction of these two photobioreactors. The photobioreactor which achieved 27.8 g/m2/day was made up of a clear glass whereas the novel tubular photobioreactor was made up of a PVC tubing. PVC tubes tend to change from clear to a milky colour after a certain period when it is used at higher temperature and pH values hence blocks a certain amount of light. Therefore the main recommendation in this study is to use a PVC tubing with a longer life span when used at a higher temperature and pH values.
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.
Correlation of saliva and serum antibody titre response to pneumococcal capsular polysaccharides
(Vaal University of Technology, 2017-10) Madimabe, Metsekae Richard; Adrian, P. V., Dr.; Grobler, C. J., Dr.
Background: Infants and young children under the age of 2 years are vulnerable to Streptococcus pneumoniae infections, especially those who are born in developing countries. Antibiotic therapy was an effective treatment until resistance to antibiotics emerged, and then vaccines were developed to assistant with the treatment. The first successful conjugate vaccine was the 7-valent pneumococcal conjugate which resulted in a decrease in vaccine serotype IPD in infants and children below the age of two years. Objectives: The main aim of this study was to assess the saliva and serum antibody concentration response to pneumococcal capsular polysaccharides in children vaccinated and unvaccinated. Design: This is a sub-study within a retrospective analysis of a prospective cohort study on the safety and immunogenicity of 7-valent pneumococcal polysaccharide-protein conjugate vaccine (PncCV) and the immunogenicity of a H. influenzae type b conjugate vaccine (HibCV). Setting and participation: Infants aged between ≥ 4 and ≤ 10 weeks were enrolled, who only received BCG and Polio vaccine following birth. Infants were enrolled according to HIV status during routine antenatal screening in the obstetrics wards of the two hospitals, in Johannesburg and Cape Town. Measurements: Saliva IgG and IgA concentrations against pneumococcal capsular polysaccharides serotype 4, 6B, 7F, 9V, 14, 18C, 19F and 23F were quantified a by multiplex bead-based assay using the Luminex technology. Serum IgG against polysaccharides serotype 4, 6B, 7F, 9V, 14, 18C, 19F and 23F were measured by a competitive Enzyme Linked Immuno-Sorbent Assay (ELISA). Results: Post three primary vaccine doses, both serum IgG and saliva IgG and IgA antibody concentrations to vaccine serotypes were protective in children who received the vaccine. The antibody concentration in children whom did not receive the vaccine was much lower in comparison to the vaccine group in both serum and saliva to vaccine serotypes (P = 0.0001). And also high positive correlation (>0.6) was observed of IgG in serum and in saliva following vaccination. Conclusion: Pneumococcal conjugate vaccines induce pneumococcal capsular polysaccharide specific antibodies in both serum and saliva. However, there are differences between the vaccines’ ability to induce mucosal immune response and there are also serotype specific differences in the antibody concentrations and in the proportion of positive samples after a series of vaccinations. The pneumococcal conjugate vaccine in this study was able to induce mucosal immune memory: the anti-pneumococcal IgA concentrations also increased with age in the saliva of unvaccinated children.
Cytotoxic and genotoxic studies of crude extracts from the leaves, stems and roots of Tulbaghia Violacea
(Vaal University of Technology, 2017-11) Nellvecia, Madike Lerato; Ssemakalu, C. C., Dr.; Takaidza, S.; Pillay, M., Prof.
Tulbaghia violacea Harv. (wild garlic) has been used in traditional medicine in Southern Africa for the treatment of various ailments. Despite the widespread use and popularity of this medicinal plant as a herbal medicine, there is contradictory evidence regarding the safety and toxicity of the plant. The phytochemical profiling of the plant has also been neglected in research. The determination of chemical constituents present in plant material as well as the potential toxicity found in plants are preliminary steps necessary for the discovery and development of novel therapeutic agents with improved efficacy. The aim of this study was to evaluate the cytotoxic and genotoxic potential of crude extracts from the leaves, stems and roots of T. violacea. This was performed in vitro using aqueous and ethanol extracts of the leaves, stems and roots. The aim of the study was achieved by three major objectives; (1) to identify the active phytocompounds present in the leaves, stems and roots, (2) to assess the cytotoxicity using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) cell proliferation assay, and (3) to evaluate the genotoxic potential of the leaf, stem and root water extracts using the Allium cepa assay. A total of 14 phytochemicals were each extracted separately with distilled water and 70% ethanol by maceration from the leaves, stem and roots of T. violacea. The results of the qualitative phytochemical analysis showed that pharmacologically active compounds such as tannins, terpenoids, flavonoids, saponins, proteins, steroids, cardiac glycosides, phenols and coumarins were present in some organs of T. violacea. However, phlobatannins, leucoanthocyanins, alkaloids, carbohydrates and anthocyanins were absent in all plant parts. Overall, the leaves of the plant contained more active compounds than those present in the stems and roots when both water and 70% ethanol were used as the extractants. The quantitative phytochemical analysis for the Total Flavonoids Content (TFC) and Total Phenolic Contents (TPC) was also assessed. The water (0.027 mg/g) and 70% ethanol (0.053 mg/g) were most effective in extracting flavonoids from the leaves while the least amounts were obtained from the stems and roots. This observation was similar to the TFC were the water extracts of the leaves were the most effective in extracting phenols followed by the stems and roots. The MTT assay was conducted using two cell lines RAW 264.7 and C2C12. The experiment was conducted in triplicates for the leaf, stem and root extracts (water and ethanol) of T. violacea. The experimental design employed a 23 factorial design where three independent variables (concentration, incubation time and type of extracts) were selected using two levels for each variable (high (+) and low (-)). The results illustrated that both the water and ethanol extracts only showed a significant reduction in the number of viable cells at the concentration higher than 250 μg/ml treatment for both RAW 264.7 and C2C12 cells. The ethanol extracts from the leaves, stems and roots were found to be toxic towards the RAW 264.7 cells even at lower concentrations at both 24 and 48 h incubation periods (% cell viability < 50%). The water extracts were non-toxic to RAW 264.7 cells except for the water stem extract which showed toxicity after 48 h incubation (IC50 = 9.475 (4.061 to 23.39)). For the C2C12 cells, the lowest potent toxic concentration was 250 μg/ml for the ethanol extract of the stem after 48 h incubation. Overall, the T. violacea plant extracts were non-toxic as percentage cell viability greater than 50% was noted for both extraction solvents in all the plant parts of T. violacea. No cytotoxic activity was observed in all T. violacea plant parts with the C2C12 cell line (IC50 > 30 μg/ml). For the Allium cepa assay, only the water crude extracts of the leaves, stems and roots of T. violacea were used. A similar trend of potent genotoxic activity in the water stem extracts compared to the leaf and root extracts at the concentration ranges studied. Similar to the MTT assay, it is clear from the study that at higher concentrations, the water crude extracts from the leaves, stems and roots of T. violacea is toxic. From this study, it can be concluded that the extraction of compounds using water is more efficient than using ethanol. Overall, the T. violacea leaf extracts extracted the most phytocompounds and showed the highest percentage of viable cells as well as desirable IC50 values. However, preparation of herbal remedies using T. violacea plant extracts should be done with caution due to their possible genotoxic and cytotoxic potential at higher concentrations. This study raises a need to further conduct in vivo cytogenetic studies to ascertain the possible toxic effects of T. violacea crude extracts.
Determining the effectiveness of water treatment process barriers for the removal of viruses in drinking water
(2018) Setlhare, Khomotso Charity; Leat, N, Dr.; Pillay, M, Prof.; Ssemakalu, C. C., Dr.
The presence of enteric viruses in drinking water poses a health risk to consumers. It is therefore very important for drinking water suppliers to provide water that is pathogen free and fit for human consumption. This can be achieved by an effective water treatment system that ensures the safety of water from the treatment plant until the water reaches the consumer. This study assessed the ability of a conventional water treatment system to remove viruses. The system consisted of three unit processes, namely, clarification, sand filtration and disinfection. These processes were simulated on a bench-scale to determine the effectiveness of each one at removing viruses. Clarification was conducted using a Phipps and Bird jar testing system and three different chemical treatments: (i) Polyelectrolyte (SUDFLOC 3835), (ii) a combination of lime and activated silica and (iii) a combination of lime, activated silica and ferric chloride. Sand filtration was simulated using a Phipps and Bird column filtration system. Disinfection was conducted using free chlorine. The findings from this study showed that the removal or inactivation of viruses increased with an increase in the concentration of chemicals added. For clarification, the combination of lime, activated silica and ferric chloride was the most effective treatment for the removal or inactivation of viruses. Sand filtration was found to be ineffective for the removal of viruses. Disinfection was shown to be the most effective process for the removal or inactivation of viruses. While clarification, sand filtration and disinfection did not remove or inactivate viruses equally, the entire treatment chain is still essential. This is because even if a barrier does not directly remove viruses it ensures that subsequent processes can function effectively. Overall the treatment processes should not be considered as discrete barriers but rather an integrated system that must function throughout to avoid a risk to customers.

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