Theses and Dissertations

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Assessment of the desalination potentials of some algal/cyanobacterial strains isolated from Buffalo River in the Eastern Cape Province, South Africa.
(University of Fort Hare, 2024) Mnikana-Cibi, Yolanda Blessings; Okoh, A.I.
Water scarcity is a global disaster, especially in arid and semi-arid countries including South Africa. When compared to the global average, South Africa's average rainfall is very low. Furthermore, freshwater scarcity is expected to worsen over the next few decades as the human population and demands grow, and the development of sustainable, energy-efficient desalination technologies could make about 97% of the water in the oceans become a useable substitute. Biodesalination using cyanobacteria is an emerging approach that could be used to achieve this feat. As a source of nutrition for growth, algae and cyanobacteria absorb ions that have a negative impact on water quality, and cyanobacteria can grow in high salt concentrations while producing a low-salt environment. Both biosorption and bioaccumulation contribute to salt removal, with biosorption having the highest salt removal efficacy. This study explores the potential of desalination potential of freshwater and marine algal/cyanobacterial strains isolated from the Buffalo River in the Eastern Cape Province, South Africa. It supports sustainable development goals related to clean water, climate action, and environmental conservation. The standard culture-based method was used for the isolation of presumptive algal and cyanobacterial strains from water collected from the Buffalo River. The presumptive cyanobacterial isolates with desalination potential were then sent to Inqaba Biotech for identification. Identification was done based on Next-generation sequencing (NGS). In total, 25 species were identified, comprising 12 cyanobacteria (Anabaena sp., MAG: Pseudanabaena, Pseudanabaena sp., Nostoc minutum, Synechococcus lividus, Hydrococcus rivularis, Spirulina subsalsa, Chroococcus sp.,Cyanobacterium aponinum, MAG: Dolichospermum, Dolichospermum sp., Chamaesiphon minutus,), diatom (Nitzschia inconspicua), and 12 green algae (Coelastrella sp., Chlamydomonas sp., Chlorella vulgaris, Monoraphidium sp., Parachlorella kessleri, Chlorella sorokiniana, Tetradesmus obliquus, Monoraphidium sp., Picochlorum costavermella, Stichococcus bacillaris, Haematococcus lacustris) isolated from the buffalo River. Out of the 20 screened isolates, 5 non-axenic (25%) strains exhibited biodesalination potential of more than 40% salinity reduction. These 5 non-axenic cultures named: 3 Wimpy, 12 Grinika, 14 Grinika, 15 Ndevana, 20 Buffalo Pass were further investigated for optimisation of culture conditions for high algal biomass to improve the efficiency of the algae/cyanobacteria biodesalination. The optimum parameters investigated included carbon source, nitrogen source, pH, light source, and effects of different salinity concentration. Salinity reduction was achieved by up to 92% salinity reduction at salt concentrations of 20 ppt seawater. Both sucrose and nitrates were found to be the best carbon and nitrogen sources under high salinity conditions yielding 55.5% and 50.0 % respectively at the regulated optimum parameters of shaker speed (180 rpm), temperature (30 ± 3 °C), 40 Watts cool white, fluorescent bulbs, and pH 7.5. To prevent the release of salt and cyanotoxins produced by cyanobacteria reverting into the desalinated water, membrane filtration was used to safely separate and recover algal and cyanobacteria strains from desalinated water. Removal of cells from the desalinated water resulted in a significant salinity reduction. To address public health and safety concerns, an in vitro toxicology study was conducted using the CyQUANT LDH Cytotoxicity Assay Kit to assess the potential risks associated with desalinated water, which turned out to be negative. The use of cyanobacteria and photosynthetic organisms in the desalination of seawater has been proven in this study. Algal strains have been shown to produce significant biomass in seawater while at the same time reducing the salt content. Using optimal conditions for algal/cyanobacterial growth, such as light, carbon, and nitrogen sources enables cyanobacteria to grow at high densities and enables future biological processes for sustainable water treatment applications. However, energy sources were a major challenge in this study. Alternative methods should be investigated for the supply of artificial energy sources. Equally, more stringent monitoring of physiological changes caused by ionic stress should be investigated. Understanding how salinity affects cell growth and viability would enable scaling up cyanobacterial growth to meet the demands of water treatment scenarios.
Genetic stock assessment of the Blue Bream, Pachymetopon aeneum (Pisces: Sparidae), a South African linefishery species, using microsatellite markers.
(University of Fort Hare, 2024-07) Yokwana, Sibusiso; Gouws, G.; Vine, N.G.
The blue bream (Pachymetopon aeneum) is a valuable species in South Africa's line fishery sector, necessitating effective management to ensure its preservation. This study utilised eight microsatellite markers to analyse the genetic diversity and structure of blue bream populations, providing insights to inform better management strategies. Microsatellites were selected for their effectiveness in assessing genetic diversity within and between populations below the species level. Given the species' vulnerability to overexploitation due to slow growth, late maturity, and protogynous hermaphroditism a comprehensive genetic stock assessment is critical. Samples were collected from 27 locations across the species' range between 2009 and 2015 by DAFF, fishing observers, and the Oceanographic Research Institute. These samples, recorded with details such as location, date, size, weight, and reproductive stage, were grouped into seven regions for robust analyses. This study aimed to determine the genetic diversity and structure of P. aeneum populations. DNA was extracted from samples, and eight microsatellite loci were amplified using fluorescently-dyed primers. Fragment analysis was conducted using the SANGER sequencer, and the fragment lengths were scored using the GeneMapper software, with quality control performed in Microchecker. Hardy-Weinberg equilibrium was assessed using Arlequin 3.5. Genetic diversity and population structure analyses were performed with GenAlex 6.5, Arlequin, and STRUCTURE 2.3.4. The results indicated high levels of genetic diversity and low inbreeding, suggesting random mating across the distribution range. AMOVA, PCoA, and Bayesian assignment identified two subpopulations. Relatedness and population assignment analyses supported these findings. Isolation by distance analysis showed no significant association between pairwise genetic differentiation and geographic distance. The evidence of two subpopulations suggests that management strategies for P. aeneum should be reconsidered. Further investigation with a larger sample size is recommended. Additionally, fishing trends should be monitored, as excessive local harvesting could impact the entire stock.
Factors influencing the larval settlement of abalone (Gastropoda: Haliotis midae): considerations for stock enhancement and ranching.
(University of Fort Hare, 2024-10) Mzozo, Ziyanda Brightness; Vine, N.G.
The South African wild abalone, Haliotis midae, is essential for critical commercial fisheries; however, the population has declined significantly over the past four decades due to overfishing and poaching. This thesis was developed in response to the decline of wild abalone stocks and the high costs associated with seeding juvenile abalone to help improve the wild population. Concerns over these costs have led to increased interest in seeding abalone larvae, as millions can be produced in a basic hatchery, resulting in lower expenses. A series of hatchery-based and ocean-seeding experiments were conducted to establish baseline methods and make recommendations for enhancing or ranching competent H. midae larvae. In one hatchery experiment, the impact of temperature on the development and settlement of H. midae larvae was assessed. The results indicated that both larval development and settlement improved with every 2°C increase in temperature within the range of 14°C to 22°C. Therefore, it is recommended that larvae be seeded at temperatures between 20°C and 22°C to potentially increase settlement rates and shorten the settlement period. The study also investigated the effectiveness of chemical and biological cues in improving the settlement and metamorphosis of competent H. midae larvae before their release into the ocean. It was found that settlement cues were only effective when water flow was turned off, and the highest settlement rates occurred with a combination of potassium chloride (10 mM KCl) and biological cues from Nitzschia sp. diatoms. However, all treatments exhibited lower settlement responses when water flow was present. This suggests that chemical cues may not be effective if larvae do not receive them until they are fully settled. Continuous exposure to an inducer until metamorphosis, achievable in a hatchery but not in the ocean, did enhance settlement. This finding highlights the potential for application within the hatchery environment. Tagging released larvae is fundamental for evaluating the success of a seeding program. In a hatchery experiment, bright and persistent epifluorescence marks were successfully induced in H. midae shells using calcein (25 mg.L-1 for 24 hours) without negatively affecting the survival or settlement of the larvae post-immersion. To evaluate the viability of using abalone seeding as a tool for ranching and stock enhancement, competent, fluorescent-stained H. midae larvae were released at three sites less than two meters apart in Noordhoek (Gqeberha) along the Eastern Cape coastline. Although the density of the released larvae (2.22±1.09 m²) was lower than that of wild abalone (37.96±7.45 m²), the growth rate of the released abalone was not hindered. Results from this pilot study demonstrate that releasing hatchery-reared H. midae larvae has the potential to support abalone ranching and enhance the wild abalone population along the Eastern Cape coastline. While the technique requires further refinement, these findings provide a foundational methodology for implementing the release of H. midae larvae in this region.
Incidence and diversity of SARS-CoV-2 from wastewater samples recovered from selected municipal wastewater treatment facilities in Buffalo City Metropolitan Municipality, Eastern Cape Province, South Africa.
(University of Fort Hare, 2024) Mayoyo, Okuhle; Okoh, A.I.; Msolo, L.
Wastewater surveillance has gained traction since the Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) induced global health crisis. SARS-CoV-2 is an enveloped Betacoronavirus that has evolved into numerous variants. The virus and its genomic variants contributed to COVID-19 transmission dynamics, disease severity, immune-response escape, and economic collapse in developing countries. This study elucidated the incidence and diversity of SARS-CoV-2 in municipal wastewaters in the Buffalo City Metropolitan Municipality (BCMM). Wastewater influent samples were aseptically collected monthly from ten wastewater treatment facilities in the municipality over a 12-month period from June 2021 to June 2022 to represent the four seasons of autumn, spring, summer and winter in South Africa. A total of 494 wastewater-influent samples were collected, and the total viral RNA was extracted from the samples following the standard operating protocols. The Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) was utilised to screen for the nucleocapsid phosphoprotein-inducing gene of SARS-CoV-2. Subsequently, the TaqMan SARS-CoV-2 mutation panel genotyping-molecular-specific RT-PCR assay, targeting the single nucleotide polymorphisms (SNPs) (delH69V70, E484K, K417N, L452R, N501Y, P681H and P681R), was used to rapidly detect SARS-CoV-2 variants with the panel related to the B.1617.2 and the B.1.1.529 lineages of SARS-CoV-2 variants. About 71% (n = 351) of the 494 samples were positive for the nucleocapsid gene, indicating SARS-CoV-2 presence. The total viral load retrieved from all study sites in winter (2021) was a total concentration of 22.08 × 104 GC/mL, spring (2021) 13.09 × 104 GC/mL, summer (2021-2022) 7.61 × 104 GC/mL and autumn (2022) 29.30 × 104 GC/mL. The circulation of variants of concern (VOC) was ascertained as 93% of SARS-CoV-2 samples (n = 45) with viral loads ≥ 1000 GC/mL were positive for the target SNPs. The predominant amino acid mutation was the N501Y (22%), shared between Alpha (B.1.17), Beta (B.1.33, B.1.351) and Omicron lineages. The geospatial analysis revealed the EB WWTP, a combined sewer network, as the diverse SARS-CoV-2 genome hotspot. Nevertheless, the spike protein mutations linked to VOCs were circulating in other areas. Overall, a direct correlation was observed between SARS-CoV-2 signals in wastewater, VOC-linked SNPs and clinical cases in the region. A significant spike in all three factors was observed in September 2021, during the third epidemic. VOC Delta (B.1.617.2) was the predominant variant during this period. Furthermore, a more significant peak was in SARS-CoV-2 with a total viral load exceeding 20 × 104 GC/mL, L452R, N501Y and P681H were detected more frequently during this epidemic driven by BA.4 & BA.5. This finding emphasises the significance of wastewater-based epidemiology as a non-invasive populationbased surveillance strategy to monitor circulating pathogens and diseases. Quantitative Microbial Risk Assessment (QMRA) model was carried out to evaluate the potential occupational health risks of exposure to SARS-CoV-2 amongst wastewater treatment plant workers at different exposure scenarios. As expected, the P(i) values for wastewater treatment plant (WWTP) operators working for 6 hours without personal protective equipment (PPE) and inadvertently exposed to up to 20 mL of contaminated bioaerosol, droplets and fomites were 35.9 × 10-2 (winter 2021), 5.1 × 10-2 (spring 2021), 6.8 × 10-2 (summer 2021-2022), and 18.2 × 10-2 (autumn 2022). There was no significant correlation between the seasons and the potential health risks (p ≥ 0.05). Therefore, the potential health risks associated with the incidence of the SARS-CoV-2 genome in wastewater across the epidemiological seasons in the study WWTPs are low at a significance level of 95%. Nevertheless, the controversies in the literature regarding the viability of SARS-CoV-2 in the wastewater milieu accentuate the coronavirus as a public health concern and demand stringent precautionary measures in occupational settings. Irrevocably, the incidence of the SARS-CoV-2 genome in the municipal wastewaters of the BCCM is an indicator of the circulation of the virus in the metropolitan. The implication of occupational and environmental safety protocols is crucial to curb the dissemination of the pathogen, and wastewater-based epidemiology coupled with QMRA are imperative approaches that could be continuously employed as complementary tools for clinical surveillance. This surveillance strategy for communicable diseases is imperative for reaching the 2030 Agenda for Sustainable Development.
Phytochemical screening and thin layer chromatographic profiling of aloe vera (l) burn. f growing in South Africa
(University of Fort Hare, 2013) Dubeni, Zimasa Busisiwe; Afolayan, A J
The chemical profiling, characterization of Aloe products and phytochemical properties of Aloe vera were studied. The adulteration of commercial products derived from medicinal plants has been a major muddle for both the society and the pharmaceuticalindustry. Economically motivated adulteration includes the potential for contaminated, sub‐potent or counterfeit medication to enter the supply chain at several levels, from the production of raw ingredients through to the point of retail sale. Darwin’s theory of evolution states that, species undergo genetic variation with time to adapt to environmental changes. Therefore, the same species growing in widely different habitats may drift from the original genetic makeup as a mechanism of adaptation and that may result in them having different chemical profiles. Therefore this study aimed at investigating the phytochemical properties of Aloe vera growing in South Africa. Also, this study aims to utilize Thin Layer Chromatography to profile this plant, as well as use Infra Red spectroscopy to characterize commercial Aloe vera products. A large quantity of Aloe vera plant was collected from AloeWay, Iphofolo Game Farm, Polokwane in the Limpopo province of South Africa. The identity of the plant was confirmedrom literature and authenticated by Professor DS Grierson of Botany Department, University of Fort Hare, Alice. The plant leaves were divided into two portions. One portion was extracted fresh while the other was cut into pieces and oven dried at 400C then and milled to a homogenous powder once dried completely. The phytochemical composition of the gel and leaf extracts revealed the presence of alkaloids, flavonoids, saponins, tannins and phenols at different concentrations. Results showed that the dry plant material yielded more phytochemicals than the fresh plant material. In particular, it was found that the acetone extract showed much more amounts ofphychemicals than the dichloromethane and aqueous extracts. The percentage compositions of phenols (71.86), flavonols (36.61), proanthocyanidins (82.71), saponins (37.73) and alkaloids (13.29) were significantly high in the acetone extract, followed by the dichloromthane extract with values of 46.85, 37.73, 49.51, 89.0 and 11.11 respectively, while the least composition was found in the aqueous extract. Furthermore, flavonoids were somewhat high in composition in both the aqueous extract of the dried and of the fresh plant material while others were very low. Tannins levels were significantly very low in all the solvent extracts. It was found that the acetone extract showed great amounts of phytochemicals than dichloromethane and aqueous extracts. Since A. vera is used in the treatment of different ailments such as skin wounds and abrasions, eczema, constipation, rheumatoid arthritis etc, the medicinal uses of this plant could be associated to such analysed bioactive compounds. Acetone, hexane, ethanol, water and dichloromethane were used to extract the Aloe vera leaf and the best solvent extract was determined. Thin layer chromatography was used to profile the leaf extracts with the aim of documenting the main phytochemicals present in the Aloe vera growing in South Africa. The best spraying reagent was determined. Fourier transform infrared spectrophotometer was used to validate the presence of Aloe vera ingredients in commercial products. The yield extraction ability of the solvent was the order: water>ethanol> hexane >dichloromethane and acetone for the dry portion. However, for the plant extracted fresh, the order of yield produced was ethanol-acetone-dichloromethane > and water. The different solvent systems separated the compounds differently. Hexane: acetone: ethanol (20 : 5: 2) and Benzene: ethanol: ammonium (80): ethanol (10): ammonium solvent systems were noted to be the best mobile phase as they gave the best separation compared to other systems.EMW [ethyl acetate (81): methanol (11): water (8)] showed better separation than the other two separating solvent systems. Vanillin- sulphuric acid spray was seen to be the best spraying reagent as compared to vanillin- phosphoric acid. Fourier transform infrared spectrophotometer validated the presence aloe ingredients in aloe vera commercial products.
Development of conservation methods for gunnera perpensa l.: an overexploited medicinal plant in the Eastern Cape, South Africa
(University of Fort Hare, 2014) Chigor, Chinyere Blessing; Afolayan, A J
South Africa, many plants which are used in traditional medicines are collected from wild populations. The high demand for trade and use of these medicinal plants place an enormous pressure on their natural populations, especially because they are indiscriminately harvested. The most affected of these plant species are those harvested from their underground parts, among which is Gunnera perpensa L. Gunnera perpensa is of considerable ethnobotanical interest in traditional medicine because of its wide usage. The rhizomes are widely used and indiscriminately collected in large quantities from the wild to meet the ever increasing demand in traditional medicine markets. As a result, this valuable medicinal plant species is being endangered. According to the Red List of South African Plants, the conservation status of G. perpensa has been listed as ‘declining’. The ethnobotanical survey conducted as part of this research confirms the plant species as threatened. It is, therefore, important to develop propagation and conservation strategies for this medicinal plant. Clonal propagation of G. perpensa was conducted using varying lengths of the rhizome (1, 2, 3, 4 and 5 cm) segments as propagules. While regeneration was possible with all the rhizome lengths, most of the growth parameters were significantly higher in the 5 cm rhizomes than the other rhizome segments. The appropriate planting depth for the rhizomes was also determined and 4 or 5 cm planting depths were found appropriate. No significant difference was observed in the growth parameters amongst the planting depths; nevertheless, 4 cm depth gave higher growth and yield. The results of this study show that regenerating G. perpensa.
The allelopathic potential of Arctotis Arctotoides (L.f.) O. Hoffm on some vegetables
(University of Fort Hare, 2012) Badmus, Abimbola Adesile; Afolayan, A J
Laboratory and greenhouse experiments were conducted to evaluate the allelopathic effects of the extracts and residue Arctotis arctotoides (L.f.) O. Hoffm on selected vegetable crops. The study aimed to address the following specific objectives to (i) examine the ultra structures of the leaf of A. arctotoides using the Scanning Electron Microscope (SEM), (ii) carry out comprehensive qualitative and quantitative phytochemical analysis of the root and shoot materials of the plant, (iii) investigate the allelopathic activities of the root and shoot aqueous extracts of A. arctotoides at concentrations of 10, 8, 6, 4 and 2 mg/ml on germination, radicle and plumule growth of cabbage, carrot, tomato and spinach, (iv) evaluate the inhibitory effects of the dried shoot residue of the plant at 10, 20 and 40 g kg-3 of soil (treatments B, C and D) and the control (treatment A) on the morphology, growth and chlorophyll pigment content of tomato and cabbage transplants at 1, 2, 3 and 4 weeks after transplanting and (v) assess the effects of the dried shoot residue of A. arctotoides on the yield, nutrient uptake by the leaves of tomato and cabbage at 4 and 12 weeks after transplanting. Finally, to analyze the residual mineral content of the soils with tomato and cabbage transplants at 12 weeks after transplanting. The the SEM revealed that anisocytic stomata and glandular trichomes (GTs) were numerous on the abaxial than the adaxial surfaces of A. arctotoides. The non glandular trichomes (NGTs) were also present on both surfaces but lesser on the abaxial. Morphologically, the GTs were peltate, uniseriate and globular head while the NGTs were cylindrical and filamentous with variable number of cells at the basal portion. The energy dispersive X-ray spectroscopy of some crystals showed that Na+ Mg2+ and Ca2+ were the major constituents of the crystal deposit found around the GTs and stomata. The results of the phytochemical composition of the root and shoot extracts of A. arctotoides confirmed the occurrence of alkaloids, flavonoids, phenolics, saponnins, tannins and triterpenes as the common constituents. In addition, cardiac glycosides and steroids were also detected in the shoot of the A. arctotoides. Quantitative estimation of the chemical constituents of the crude extracts further revealed that the alkaloid content in the root higher (0.97 percent) than the shoot (0.64 percent). The quantity of flavonoids detected in the shoot (1.02 percent) was more than that observed in the root (0.35 percent). Others (phenolics and tannins) were marginal in the two plant parts. The results of the inhibitory effects of the root and shoot aqueous extract at the varying concentrations showed that root extract at 10 mg/ml considerably reduced the germination of cabbage, carrot, tomato and spinach seeds by 84.0, 83.2, 72.8 and 37.4 percent respectively. Incubation of the shoot extract at the same concentration resulted in 100 percent inhibition of cabbage and carrot seed germination whereas those of tomato and spinach were suppressed by 91.5 and 61.2 percent respectively. The two extracts at the varying concentrations also had a significant reduction on the radicle and plumule growth of the four vegetables. Addition of the shoot residue to the soil showed massive chlorosis, necrotic lesions and wilting of tomato and cabbage leaves under treatments C and D at 2 weeks after transplanting. The number of leaves, leaf area, dry shoot and root weight of the two vegetables grown in the amended soils were also drastically reduced. The inhibition percentages due to the addition of the three concentrations of A. arctotoides dried shoot residue on the dry shoot weight at 4 weeks after transplanting were 38.6, 45.5 and 70.3. for tomato and 57.5, 73.3 and 87.5 percent for cabbage. Similarly, the declines in the dry root weight of 61.3, 82.9.4 and 83.4 percent for tomato as well as 53.1, 54.7 and 67.2 percent for cabbages were recorded for the two vegetables under treatment B, C and D during the period. The results further showed that the dry fruit yield and shoot weight of tomato under the treatments B, C and D decreased with increase in shoot residue concentrations of A. arctotoides. Relative to treatment A, no significant differences were recorded in the dry head weight of cabbage under the residue treated groups. The reductions in the fruit yield and fresh head weight caused by treatments C and D were 37.2 and 84.8 percent for tomato and 30.9 and 72.4 percent for cabbage. The findings on the mineral contents in the leaves of the two vegetables revealed significant differences in the uptake of N, Mg, Na, Cu and Fe by tomato leaves. The concentrations of N, K, Na and Zn in cabbage leaves also differed. However, the P content was relatively constant in the leaves of the two vegetables at 4 and 12 weeks after transplanting. At 12 weeks after transplanting, the Fe content in soils with tomato and cabbage treatments C and D was greatly enhanced in comparison with the other nutrients. The residual N, P and Zn detected in soils planted to cabbage were similarly equal among all the groups including the control. Thus, under the greenhouse experiment, Arctotis arctotoides (L.f) O. Hoffm has been shown to contain some phytotoxic chemical compounds in its root and shoot materials. The compounds either singly or collectively have demonstrated some inhibitory potentials on the germination, growth and yields of cabbage, carrot, tomato and spinach evaluated in this study.
Validating the use of Hippobromus pauciflorus (L.f) Radlk for the treatment of eye infections in the Eastern Cape South Africa
(University of Fort Hare, 2010) Pendota, Srinivasa Chary; Afolayan, A J; Grierson, D S
An ethnobotanical survey of plants used for the treatment of eye infections was carried out in the Eastern Cape Province of South Africa. The study revealed that 12 plant species are commonly used with Hippobromus pauciflorus (L.f) Radlk being the most frequent. The crude extracts of the leaves, stem bark and roots of the plant were investigated for antimicrobial activity against 10 bacterial and four fungal strains. The methanol extracts of the plant parts were the most active and showed appreciable activity against Gram-positive and Gram-negative bacteria. The methanol extracts of the leaves and stem bark inhibited the growth of fungi with activities ranging from 78.70 percent to 100 percent on Aspergillus niger and Penicillium notatum. The acetone extracts of the leaves and stem bark were active against A. niger (51.76 percent) and P. notatum (77.22 percent). The aqueous extract of Hippobromus pauciflorus leaves at 50, 100 and 200 mg/kg body weight doses were evaluated for anti-inflammatory, analgesic and antipyretic activities in male Wistar rats. Anti-inflammatory activity was studied by using carrageenan-and histamine induced oedema right hind paw volume while the analgesic effect was evaluated using formalin-induced pain and tail flick nociception response. The brewer’s yeast-induced pyrexia model was used for the antipyretic investigation. The extract at all the doses used significantly inhibited both the carrageenan- and histamine-induced inflammation in a manner that was not dose dependent. The extract reduced the formalin-induced pain licking as well as prolonged the reaction time in the tail flick-induced pain. The effects of the aqueous extract of the plant leaves at 50, 100 and 200 mg/kg body weight doses were investigated for 14 days on some biochemical parameters of male Wistar rats. The extract at all the doses tested did not significantly alter the levels of white blood cells, red blood cells, mean corpuscular volume, platelets, neutrophils, monocytes, lymphocytes and large unstained cells. While the levels of haemoglobin, packed cell volume and basophils increased at specific doses, those of mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and eosinophils decreased. In vitro antiviral activities of the plant against herpes simplex virus type 1(HSV-1) and coxsakie virus B6 were investigated. Cytotoxicity was evaluated by MTT assay in Vero cells. At concentrations ranging from 165-270/μg/ml there was significant antiviral activity against HSV-1, but toxicity was also noted. There was no antiviral activity against coxsakie virus B6. Bioactivity-guided fractionation of the leaves of H. pauciflorus yielded three known compounds. From the ethyl acetate fraction, two compounds were isolated (epicatechin and -sitosterol ) and from the n- hexane fractions, one compound (lupeol) was isolated. They were isolated and identified using various techniques. The antimicrobial, anti-inflammatory, analgesic and anti-pyretic activity of these compounds have been reported in literature. The structure and distribution of foliar appendages on the leaves of H. pauciflorus were examined by scanning electron microscope. The leaves have long unicellular nonglandular trichomes which were distributed over the mid rib and densely populated at the edges of the adaxial and abaxial surfaces. In general, the experiments and tests conducted in this study appear to have justified the use of Hippobromus pauciflorus for the treatment of eye infections and make a substantial contribution to the knowledge base of the use of herbal medicine for the treatment of the eye infections.
Effect of processing on the efficacy and safety of Solanum Aculeastrum Dunal berries
(University of Fort Hare, 2009) Aboyade, Oluwaseyi Mayode; Folayan, A J; Grierson, D S
General Abstract There has been a steady increase in the use of medicinal plants in the last two decades in both developed and developing countries for prevention, management and treatment of diseases. This increase has been due to reasons such as ease of access, better cultural acceptability and compatibility, cost effectiveness and also the bid to “go natural”. Unfortunately, the notion that herbal medicines are relatively safe because they are natural has led to serious and potentially fatal consequences in phytotherapy. The lack of rigorous research to prove the effectiveness and safety of many medicinal plants is of great concern to the health care system. This thesis therefore addresses not just the efficacy, but also the safety of the extracts of the berry of Solanum aculeastrum - a medicinal plant used, among other things, for the treatment of breast cancer in the Eastern Cape Province of South Africa. Particular attention was paid to the possible effect of different processing methods of the berry extracts on inflammation, cytotoxicity, and toxicity. In studying the comparative effects of various processing methods, four different preparations of the extracts were investigated. These include fresh, dried, boiled fresh, and boiled dried berries. While the effect of processing on the anti-inflammatory properties of the extracts was not dose dependent, the percentage reduction in inflammation was highly significant and more prominent in both concentrations of the boiled fresh berries than the reference drug, indomethacin. Furthermore, the four extracts varied in their ability to act either centrally or peripherally in their effect on pain. Assessment of the analgesic response using the formalin test showed that, at both concentrations tested, none of the extracts inhibited the first phase of the formalin test. Furthermore, it was observed that boiling had differing results on the activity of the iii fresh and dried extracts. While boiling of the dried berries reduced pain in the rats, the opposite trend was observed with the boiled fresh berries. Results of the influence of processing of the berries on cytotoxicity indicated that the extracts are potent inhibitors of human breast, cervical and colonic carcinoma cells and the non-cancerous cells (both the actively dividing and confluent Chang liver cells). Although, in terms of relative potency, the fresh berries appeared to be the most active of the extracts, processing of the berries caused an increase in apoptotic cells and a subsequent decrease in the necrotic cells. The effect of processing on the safety of the berry of S. aculeastrum on the rats fed for 28 days was also investigated. The various doses (1, 10 and 25 mg/kg body weight) of all the four extracts did not alter the activity and the weight of the animals throughout the period of treatment. A reduction in organ to body weight ratio of the heart, kidney, liver and spleen was observed in all the extracts. Regarding the haematological parameters, different patterns of effect were observed between the extracts and within the treated doses. The observed alterations in the biochemical parameters by the various extract of Solanum aculeastrum berries at all the doses may have consequential effects on the normal functioning of these organs. In conclusion, this study has shown that there is some justification for the traditional use of the berries of Solanum aculeastrum in the treatment of inflammation related ailments and cancer. However, the medicinal use of the plant also poses considerable health risks. Investigation conducted into the plant’s safety revealed that the berry extracts were nephrotoxic, hepatotoxic, haematotoxic and at higher doses, fatal. Another concern with regard to the plant’s safety is the non-selectivity of its extracts in the inhibition of carcinoma, actively dividing and un-dividing cells. Assessment of the effect of the processing on the berry’s efficacy and safety as herbal iv remedy produced mixed results. On the one hand, processing seemed to improve the extract’s anti-inflammatory and analgesic activity, while reducing its cytotoxic potential. On the other hand, a reduction in the toxicity was observed on the processed extracts compared to the fresh ones. This may be an indication that processing has an overall beneficial effect on the medicinal properties of the plant and should thus be considered as a method of making the berries of Solanum aculeastrum safer for use as a herbal remedy.
Heavy metal content absorption and medicinal potential of Egeria densa (Planch.) Casp
(University of Fort Hare, 2013) Mgobozi, Vuyokazi; Afolayan, A J
The contamination of heavy metals in the environment is a looming concern worldwide. Egeria densa (Planch) (Submerged aquatic plant) from two ponds: Site A with co-ordinates (32º 48’22.04”S; 26°48’58.79” E) and Site B with co-ordinates (32°48’33.25”S; 26°48’33.25”S) in Alice (Eastern Cape) was evaluated for its ability to absorb heavy metals, phytochemical constituents, antimicrobial activity and ultra-structure using standard analytic procedures. Cadmium (Cd), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), and zinc (Zn) were measured in water, sediments and plant. The concentrations of these metal elements were determined with use of Inductively Coupled Plasma- Optical Emission Spectrometry (ICP-OES). In sediments, the heavy metals (mg/kg) decreased in the order of their average concentration as follows: Fe (40.320) > Zn (1.259) > Pb (0.564) > Mn (0.186) > Cu (0.037) in Pond 1 whereas in Pond 2 Fe (61.527) > Cd (0.999) > Mn (0.648) > Pb (0.586) > Zn (0.156) > Cu (0.045). The highest concentration of Fe was detected in both sites and Cu being the least. The concentrations of the metals in the plants sample (from Pond 1) were found in order of Mn > Pb > Cu > Fe whereas cadmium and zinc were not detected, while the concentration in Pond 2 decreases in order of Zn > Mn > Pb > Cd > Fe > Cu. In the water samples, concentrations of heavy metals (mg/L) decreased in the order of their average concentrations as follows: Pb (35.36) > Fe (3.07) > Mn (0.238) > Cu (0.104), both cadmium and zinc were below the limit of detection in Pond 1, whereas in Pond 2 the concentrations decreased as follows: Pb (13.033) >Fe (1.69) > Cu (0.270) > Mn (0.248) > Cd (0.004) and Zinc was not detected. Phytochemical analyses of the plant extracts revealed the presence of phenols, flavonoids, proanthocyanidin, flavonols, saponins, alkaloid and tannins in all the extracts (water, acetone and n-hexane). Both acetone and water extracts, showed high concentration of proanthocyanidin, while tannin was the lowest in acetone extract. Antimicrobial evaluation using, Gram positive (Staphylococcus aureus, Bacillus pumilus, Bacillus cereus, Streptococcus pyogenes, Enterococcus faecalis) and Gram negative (Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Serratia marcescens) bacteria showed negative results for all the strain, except Streptococcus pyogenes which was inhibited at MIC of 0.1 mg/ml. Scanning electron microscopy (SEM) of ultra-structure of Egeria densa, showed that certain bacteria attached to the leaf, However more work has to be done on E. densa to verify the mechanism by which it accumulates heavy metals. The study shows that E. densa has a potential of accumulating heavy metals especial Manganese in plant.
Studies on the use of essential oils for the control of Sitophilus Zeamais (Motschulsky) (Coleoptera; Curculionidae): a pest of stored maize grains
(University of Fort Hare, 2008) Odeyemi, Oluwakemi Oluwaseyi; Afolayan, A J
The common maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), a well known pest of stored-maize grain in most parts of the world, was identified as one of the major constraints of harvested maize grains in the Eastern Cape Province, South Africa. The use of plants or their products is one of the recent methods being investigated for insect pest control worldwide. Therefore, the main aim of the present study was to identify available plants in the Eastern Cape that could be used to combat the problem of Sitophilus zeamais in stored-maize grains. For the realization of the aims of this research, the following studies were carried out; a preliminary survey was conducted to obtain baseline information on the farmers’ knowledge and experience of indigenous insect pest control methods in the Eastern Cape. Also, studies on the insecticidal potential of the essential oils of some plants were investigated against the maize weevil. The quality parameters of maize grains treated with the essential oils was also studied and, using a rat model, the toxicity of the essential oils was investigated. The outcome from this study revealed that there is awareness amongst the farmers in the Eastern Cape on the use of plants or their products to control insect pests. Unfortunately, such methods are currently being neglected and the knowledge of their application was found to be eroding. Among the various essential oils screened were those from Mentha longifolia L. and Tagetes minuta L. which evoked an appreciable level of contact, fumigant and repellent toxicity on the maize weevil. Further work done to determine the effects of the oils on maize stored over a period of three months revealed that the two oils had no adverse effect on the proximate compositions and some quality parameters of the stored maize. However, the toxicological study conducted on rats showed that the oils at tested concentrations exhibited some level of toxicity. It is, therefore, suggested that the essential oils of M. longifolia and T. minuta should not be used to treat maize grains intended for human consumption.
Antifugal evaluation and phytochemical analysis of selected medicinal plants used in the treatment of fungal diseases associated with HIV infection in the Eastern Cape Province, South Africa
(University of Fort Hare, 2013) Mbeng, Wilfred Otang; Grierson, Donald S; Ndip, Roland, N
Background. As a result of the AIDS pandemic, many people areimmuno compromised andopportunistic fungal infections (OFIs) such as candidiasis are common. Despite the widespread use of medicinal plants in South Africa, there is a dearth of knowledge regarding the use of such plants in the management of these infections. This study evaluates three South African medicinal plants (Arctotis arctotoides, Pittosporum viridiflorum, and Gasteria bicolor) traditionally used in the treatment of OFIs in HIV/AIDS patients, in the Eastern Cape Province, South Africa. Materials and methods. A six-stage process of documentation, evaluation and analysis of results was conducted: (1) Selection of medicinal plants most frequently used in the treatment of OFIs through ethnomedical studies and the survey of specialised literature; (2) Collection and preparation of the extract of each plant; (3) Antifungal evaluation of the crude plant extracts. (4) Phytochemical and antioxidant evaluation of the active crude plant extracts; (5) Cytotoxicity evaluation of the bioactive extracts using the Chang liver cell line, and (6) Statistical analysis of the results. Ethnobotanical information was obtained through interviews with traditional healers and AIDS patients with the aid of semi-structured questionnaires, direct observations and by reviewing studies reported in the literature. Following the approval from the University of Fort Hare‘s Ethics Committee, 101 HIV/AIDS patients were recruited through convenience sampling into an anonymous cross-sectional questionnaire study. The agar diffusion and micro-dilution methods were used to determine the antifungal activities of the hexane, acetone and aqueous extracts of A. arctotoides, G. bicolor and P. viridiflorum against 10 opportunistic fungi.
Comparative phytochemical analyses of Aloe Ferox Mill. found in Eastern and Western Cape provinces in South Africa
(University of Fort Hare, 2013) Adams, Zanele; Afolayan, A J
Assessment of bioflocculant production by two marine bacteria isolated from the bottom sediment of marine Algoa Bay
(University of Fort Hare, 2015) Ntozonke, Ncedo; Okoh, Anthony I; Nwodo, Uchechukwu U
Bioflocculants are polymers, mostly, of microbial origin which floc out suspended particles from liquid medium. The ability of these biopolymers to remove suspended particles from solutions is termed bioflocculation, and the efficiency of flocculation activities depends on the characteristics of the flocculants. In comparison with conventionally used flocculants, bioflocculants have the advantage of being safe (no toxic effects known), biodegradable and harmlessness to the environment. The study assessed production of bioflocculant by two marine bacteria from the bottom sediment of marine environment. The 16S rDNA was used for identification, and the two bacteria species were identified as Enterococcus hirae and Bacillus thuringiensis. Factors affecting the production and activity of the bioflocculants produced by these two organisms were studied. The bacteria optimally produced bioflocculant with fructose (91.7%) and urea (91%) as sole carbon and nitrogen sources respectively. Mg2+ (87%) and Ca2+ (86%), likewise, served as best cation sources on the production of the bioflocculant at pH 5(93%). Additionally, the flocculating activity of the bioflocculant increased with the addition of Mg2+ (81%) and Na+ (81%), and the highest flocculating activity was at pH 5 of the kaolin clay. The Fourier transform infrared spectroscopy (FTIR) shows that the bioflocculant is a glycoprotein. The second bacterium (Bacillus thuringiensis) produced bioflocculant optimally when the media had mixed nitrogen sources (Urea, ammonium chloride and tryptone (67%)) and glucose (85.65%) as a sole carbon source, also Ca2+ (74.6%) was the best cation that induced the production of bioflocculant. After purification, the bioflocculant flocculated optimally in alkaline pH 12 (81%) in the presence of Mn2+ (73%) and Ca2+ (72.8%). Chemical analysis of the bioflocculant revealed it to be a polysaccharide. Both bioflocculants flocculate efficiently and can be used to replace synthetic flocculants in water treatment, wastewater, in downstream processing, and processing of food and chemicals and other industrial uses of flocculants. Challenges though (i) are to develop conditions for large scale production of the bioflocculant, (ii) to do further characterization of the both bioflocculants (iii) to assess the bioflocculants for treatments of water/wastewater, and to apply it in various industrial processes.
Isolation and characterisation of lignocellulose degrading bacteria from Tyume River in the Eastern Cape Province, South Africa
(University of Fort Hare, 2015) Tembisa, Papiyana Ayavuya; Okoh, Anthony I
This study focuses on the isolation and characterization of bacteria from lignocellulosic biomass obtained from the sediments of the Tyume River in Alice, Eastern Cape and to determine those bacterial isolates with good potential for modification and decomposition of lignocellulosic biomass for industrial application. Several bacterial isolates were recovered and screened for ability to degrade various lignocellulosic materials. Nine of the isolates were positive for lignocellulolytic activity. Four isolates were cellulase positive and six were xylanase positive. Moreover, one isolate (SB1) was positive for both xylanase and cellulase activities and showed the best hydrolysis zone on solid media. This isolate was then chosen as the best and identified molecularly. The 16S rDNA sequence analysis indicated that SB1 was a Bacillus cereus species. Factors affecting the cellulose and xylanase enzyme production by the organisms were studied. The organisms produced the enzymes maximally at earlier hours of incubation (12-30 hr) and optimally at acidic pH (3-5) and at moderate temperatures (35-45ºC). SB1 appears to hold promise in the decomposition of lignocellulosic wastes.
Assessment of the flocculating efficiency of bioflocculant produced by bacillus sp. Aemreg4 isolated from Tyhume river, Eastern Cape, South Africa
(University of Fort Hare, 2016) Ntsangani, Nozipho; Mabinya, L V; Okoh, Anthony I
Bioflocculants are flocculating substances produced by microorganisms during growth and have recently received considerable attention from researchers; due to their biodegradability, non-toxicity and lack of secondary pollution from degradation intermediates. This study evaluated the efficiency of bioflocculant produced by Bacillus sp. AEMREG4 isolated from Tyhume River. The bacterial identification was through 16S rDNA sequencing; nucleotide sequences were deposited in GenBank as Bacillus sp. AEMREG4 with an Accession number KP406729. The optimum culture conditions for bioflocculant production were an inoculum size of 4% (v/v) and starch as well as yeast extract as sole carbon and nitrogen sources respectively. The addition of CaCl2 enhanced the flocculating activity, at a wide range of pH 4-10 and the highest flocculating activity was reached at an initial pH 8 (80%). A bioflocculant yield of 0.78 g was recovered from 1 L of culture broth. The optimum flocculating activity of 78% was reached at the lowest bioflocculant dosage of 0.1 mg/ml and the presence of divalent cations (Ca2+, Mn2+ and Mg2+) as well as a trivalent cation (Al3+) enhanced flocculating activity. The purified bioflocculant retained more than 70% flocculating activity when subjected to heating at 100 °C for 1 h and maximum flocculating activity of 83% was achieved at both acidic and basic pH values of 3 and 10 respectively. Chemical analysis showed that the bioflocculant is predominantly polysaccharide. The Fourier transform infrared (FTIR) spectrum revealed the presence of carboxyl, hydroxyl and methoxyl groups as the functional moieties and the scanning electron microscopy (SEM) imaging of the purified bioflocculant showed its morphological structure as rod-shaped which contributes to its high flocculating efficiency. The high flocculation activity displayed by this bioflocculant indicates its potential suitability for industrial application.Keywords: Bioflocculant, Bacillus sp. AEMREG4, flocculating activity.
A preliminary study on the effects of elevated CO2 on aphid resistance of Tugela Dn and the population dynamics of the Russian wheat aphid (Homoptera: Aphididae), Diuraphis noxia
(University of Fort Hare, 2015) Mundondo, Daphine; Bradley, G
Food security is of major importance due to the increasing world population with 8.9 billion people expected by 2050 (Cohen, 2003). Diuraphis noxia (RWA), have caused aggravating, massive losses to wheat farmers in many areas of the world. If unchecked, RWA are able to destroy plants resulting in major economic impacts (Botha, 2013). Due to ineffective use of other control methods, the Small Grains Institute in Bethlehem, South Africa, have therefore developed resistant cultivars to the known RWA subtypes over the past decades through intensive breeding programmes (Tolmay et al., 2006). Climate change has however become a major factor threatening food security especially with the observed increase in CO2 from less than 300 ppm in pre-industrial period to the current 385 ppm and is predicted to reach 550 ppm by 2050 (IPCC, 2007; Meehl et al., 2007). Elevated CO2 concentration may affect individual species of a community hence the need to understand the wheat-aphid interactions. In this study, population growth rates and virulence of RWA SA1 at ambient (385 ppm) and elevated (450 ppm) CO2 concentration were evaluated on two wheat cultivars: Tugela Dn (resistant) and Scheepers (susceptible). Fluorescence microscopy techniques using aniline blue were used to investigate feeding related damage caused by RWA SA1 through an examination of callose deposition at the two CO2 concentration. A two-dimensional gel electrophoresis method was developed in order to determine the effect of RWA SA1 on the wheat cultivars proteome at the two CO2 concentration. Differentially expressed proteins that were up or down regulated more than two fold were identified using PDQuestTM Basic 2D Gel analysis software. Populations of RWA SA1 increased significantly on the two wheat cultivars at both CO2 concentration. Although the population growth rate for RWA SA1 on both cultivars was generally exponential at all treatments, growth at elevated CO2 concentration was noticeably faster with populations increasing 3 fold in 14 days as compared to the 2 times at ambient CO2 concentration. Hence, both cultivars provided a better quality host for RWA SA1 at 450 ppm than 385 ppm. There was no significant difference between RWA SA1 population on Tugela Dn and on Scheepers at elevated CO2 concentration on day 14 after infestation which means there was a change in the resistance mechanism in Tugela Dn at this condition. Approximately 70% of the total leaf showed chlorosis by 21 days of aphid infestation for both cultivars although the susceptible cultivar was more vulnerable. There was low callose deposition in the controls (uninfested plants) but heavy callose in infested plants due to aphid feeding. A proteomics approach was used as a pilot study to investigate whether it would be possible to identify the changes in the resistance mechanism during aphid infestation under elevated CO2 levels. The major changes in the proteome of the control group (uninfested Tugela Dn at ambient versus elevated CO2 concentration) occurred in the early events (day 1-7) in the molecular weight range of approximately 25 kDa to 55 kDa are mainly within the basic to neutral pH range. This was suggested to be a result of mechanisms to adjust to the CO2 concentration. Elevated CO2 results in instant higher photosynthetic rates and C:N ratios as well as changes in expression levels of SA-dependant defense genes (Lindroth 1995; Hughes and Bazzaz, 2001; Sun et al., 2013). Because most of these changes are directly regulated by proteins, it is expected that the most differential protein expression will occur immediately after the atmospheric changes (early events) as was shown in the study. Infested plants under elevated and ambient conditions showed that the stress conditions gave rise to differentially regulated proteins within the wheat proteome. Most changes occurred elevated CO2 levels. It can be suggested that the changes were a result of differentially regulated plant defence proteins which fall in this range (25 kDa - 80 kDa) such as peroxidases, chitinases and β-1.3-glucanases as well as protein kinases, heat-shock proteins and photosynthetic proteins. These results indicate that there has been changes in the resistance due to elevated CO2 because of the evident changes in the proteome. If so, then the results will be similar to those documented by Louw (2007) where up-regulation was due to putative storage proteins, proteins involved in photosynthesis, heat shock proteins and defense proteins. Of course, the pI value and molecular mass of the proteins and the identification of the proteins in these spots, must be determined in future work to specifically identify whether these suggestions are authentic. However, Louw (2007) also reports that the susceptible Betta wheat cultivar, displayed a defence response similar to the HR although it was unable to up-regulate specific defensive proteins against RWA infestation but proteins for broad resistance. Although the changes in the proteins in infested Tugela Dn under elevated CO2 concentration were not accurately identified, the defense mechanism is similar to that portrayed by the susceptible Betta wheat cultivar which shows that the resistance mechanism had been overcome. Because this was a pilot study and preliminary results were obtained due to limited funding and time constraints, suggestions were made on how to further develop the method to obtain statistically significant results.
Phytochemical analysis and antibacterial properties of aqueous and ethanol extracts of Brachylaena elliptica (Thurb.) dc. and Brachylaena ilicifolia (Lam.) Phill & Schweick
(University of Fort Hare, 2015) Sagbo, Idowu Jonas; Afolayan, A J; Bradley, G
Resistance of human pathogenic bacterial strains results in selective pressure against known antibiotic. However, plant derived compounds that possess antibacterial potential are currently being investigated for treatment of wound infections in diabetic patients as they are inexpensive and non-toxic. Hence, this dissertation was designed to evaluate two medicinal plants (Brachylaena elliptica and Brachylaena ilicifolia) traditionally used in the treatment of various diseases such as diabetes, and its secondary complications in diabetic patients. The in vitro antioxidant activity of both plants were evaluated using DPPH (1, 1-diphenylhydrazl), ferric reducing power, ABTS (2, 2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), NO (nitric oxide) and H2O2 (hydrogen peroxide) techniques. The antibacterial test and Minimum inhibitory concentration (MIC) was determined by agar dilution method against 5 bacteria strains (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogene, Proteus vulgaris and Proteus mirabilis) infecting wounds in diabetic patients using amoxicillin and ciprofloxacin as positive control. The phytochemical analyses were assessed using standard published methods. Identification of bioactive components in essential oils of both plants were assessed using GCMS. The aqueous and ethanol extracts of both plants were also evaluated to identify bioactive components using LC-MS. The results of the phytochemical analysis revealed the presence of phenols, tannins, flavanoids, flavanols, proanthocyanidins, saponins and alkaloids in both plants. Both plants indicated strong antioxidant activities which might be due to the presence of bioactive compounds. The aqueous and ethanol leaf extracts of both plants demonstrated appreciable broad spectrum activities against these wound pathogens with MIC ranging between 5 and 0.3 mg/ml. The GC-MS analysis of the essential oils of both plants revealed the presence of monoterpenes, oxygenated sesquiterpenes, phenolics and esters. The LC-MS analysis of the aqueous and ethanol leaf extracts of both plants showed that both plants are rich in alkaloids, terpenes, terpenoids, monoterpernoids, and flavanoids. Conclusively, this study has partially justified the ethnomedicinal use of B. elliptica and B.licifolia leaves for the treatment of various diseases, including diabetes and wound infections caused by bacteria in diabetic patients. These may be attributed to the presence of antioxidant compound such as phenols, flavanoids, saponins, tannins, alkaloids and other phytochemical compounds.
Characterization of some virulence and antibiotic resistance genes of Staphylococcus aureus isolated from cases of Bovine Mastitis in Nkonkobe Municipality, Eastern Cape Province, RSA
(University of Fort Hare, 2015) Pekana, Abongile; Green, E; Okoh, Anthony I; Ndip, R N
Staphylococcus aureus is one of the predominant causative agents of mastitis disease in dairy herds. Mastitis disease has a negative impact in the economic losses in the dairy sector across the globe. The aim of this study is to detect some of the virulence genes in the S. aureus isolated from 400 milk samples of subclinical and clinical mastitis dairy cows in Fort Hare dairy farm and Middle Drift dairy farm in Alice in the Eastern Cape province of South Africa. In addition antibiotic resistance pattern and antibiotic resistance genes were investigated. Gram-staining, oxidase test, catalase test and API Staph kit were preliminary biochemical tests used for the identification of S. aureus isolates. The MALDI-TOF-MS was also used for further identification. Polymerase chain reaction was performed of genes encoding antibiotic resistance as well as clumping (clfA), coagulase (coa) gene, toxic shock syndrome (tsst), exfoliative toxin A and B (eta and etb), and the gene segment encoding the immunoglobulin G binding region and X region of protein gene spa. A total of 20 (5%) S. aureus strains obtained from 400 milk samples from the two farms were subjected to 16 antibiotics for antibiotic susceptibility testing. In Middle Drift dairy farm 11 (5.5%) isolates were obtained from 200 samples and 9 (4.5%) isolates were obtained in Fort Hare dairy farm from 200 samples. A large percent of the isolates were resistant to penicillin G (60%), followed by trimethoprim (60%) and tetracycline (60%), trimethoprim-sulfamethaxazole (55%), telithroprim (55%) and doxycycline (45%). Most of the isolates were sensitive to several (50-85%) antibiotics. Of the twenty isolates tested 12 samples contained the penicillin antibiotic resistance gene (blaZ gene), 8 samples contained at least one aminoglycoside-modifying enzyme gene (AME gene); the (aac(6’)/aph(2’’) gene and no amplification occurred for aph(3’)-IIIa and ant(4’)-Ia) genes. In the case of the tetracycline antibiotic resistance gene (tetK and tetM), 2 samples contained tetM and a single sample contained tetK gene. No amplification was observed for the erythromycin antibiotic resistance genes (ermA, ermB, ermC, Mef and msrA). All the samples tested were negative for the expression of toxic syndrome gene (tsst), etb, and Immunoglobulin G binding region. However, amplification of the clumping factor was observed in 7 (35%) isolates of S. aureus, exfoliative toxin (eta) expressed 4(20%) isolates; coagulase gene (coa) yielded six DNA bands of six differences sizes from 16 (80%) isolates. A total of four different bands size were expressed for the spa X region from 12 (60%) isolates. The data obtained in this study suggests that poor hygienic practices and inadequate management practices are responsible for the increase in Staphylococcus aureus isolation. The high resistance of S. aureus to antibiotics and the distribution of virulence genes contribute in bovine mastitis in these farms may cause health problems in the community consuming raw milk purchased from these farms.
Isolation and molecular characterization of Bacillus cereus from cow’s raw milk
(University of Fort Hare, 2015) Lukanji, Zinathi; Ndip, R N; Green, E; Okoh, Anthony I; Ndip, R N
Bacillus cereus is a group of ubiquitous facultative anaerobic spore forming Gram-positive rods commonly found in soil. It has been detected and implicated in several contaminated food products and raw milk in dairy farms and it causes foodborne gastroenteritis by producing several toxins. This study is aimed at characterizing virulence determinants of B. cereus from cow‟s raw milk. A total of 400 raw milk samples were collected in Fort Hare Dairy Trust and Middledrift Dairy Farm; and cultured on Polymyxin pyruvate Egg-Yolk Mannitol Bromothymol Agar (PEMBA) for 48 hours at 37°C. DNA was isolated from the isolates and 16S rDNA was amplified and sent to Central Analytical Laboratory for sequencing. The gyrB gene of B. cereus was also used to confirm the identity of the isolates. Antibiotic susceptibility profiles of the isolates together with virulence genes were investigated. Multilocus Sequence typing was used to investigate the genetic relatedness of some selected isolates. Furthermore, spores of the isolates were produced, harvested and their concentrations determined. All (100%) of the isolates were identified as having a 96-99% similarity to B. cereus, B. thuringiensis and B. anthracis using sequencing; while gyrB gene was observed in all (100%) of the isolates. Three virulence genes nheA, nheB, nheC encoding for non haemolysin enterotoxin were amplified in all (100%) the isolates. All (100%) of the isolates were susceptible to doxycycline, gentamycin, tetracycline, ciprofloxacin, chloramphenicol and streptomycin. Resistance to rifampicin and penicillin G was predominant with equal rate of 100%, while susceptibility to erythromycin, clindamycin and doxycycline ranged from 60% to 100%. The selected isolates were related and are descendants of the same ancestor. All (100%) the isolates produced spores. The B. cereus isolates contain virulence genes, has multiple antibiotic drug resistance and produce spores, which poses a health risk to the public and cannot be used as probiotics.

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