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Item Electrospun carbon nanofibers as an electrochemical immunosensing platform for vibrio cholerae toxin: Aging effect of the redox probe.(ACS Publications, 2020-03-12) Celestine, Okoroike Ozoemena; Maphumulo, Tobile; Shai, Jerry L.; Ozoemena, Kenneth I.An electrochemical immunosensor for Vibrio cholerae toxin (VCT) has been developed using electrospun carbon nanofibers (CNFs) as the electrode platform. To fabricate the immunosensor, the anti-cholera toxin antibody (Ab) was covalently immobilized on the electrode platforms using the carbodiimide chemistry for the amide bond formation. Every step of the formation of the immunosensor and the subsequent binding of the VCT subunit antigen (Ag) was electrochemically interrogated. The immunosensor gave excellent reproducibility and sensitivities: limits of detection (ca. 1.2 × 10−13 g mL−1), limits of quantification (ca. 1.3 × 10−13 g mL−1), and a wide linear range for the anti-cholera detection of 8 orders of magnitude (10−13 Mto 10−5 g mL−1). One of the key findings was the enhanced sensitivity of the VCT detection using aged rather than the freshly prepared redox probe, described here as Redox Probe Aging- Induced Sensitivity Enhancement (“Redox-PrAISE”). The Redox-PrAISE was found more useful in the real application of these immunosensors, showing comparable or even better sensitivity for eight real cholera-infested water samples than the conventional clinical culture method. This immunosensor shows promise for the potential development of point-of-care diagnosis of VCT. Importantly, this study highlights the importance of considering the nature of the redox probe on the electrochemical sensing conditions when designing impedimetric immunosensors.Item Bovine serum albumin‑dependent charge‑transfer kinetics controls the electrochemical immunosensitive detection: Vibrio cholerae as a model bioanalyte.(Springer, 2021-06-09) Ozoemena, Okoroike C.; Tobechukwu J. Ehirim, Tobechukwu J.; Khawula, Tobile; Makgopa, Katlego; Shai, Leshweni J.; Ozoemena, Kenneth I.immunosensors, can impact on the sensitivity of detection when integrated with antibody (Ab) pre-encapsulated with (i) insulating polyacrylonitrile (PAN) fibre (i.e., GCE-PAN-Ab-BSA immunosensor) or (ii) conducting PAN-grafted iron (II) phthalocyanine (FePc) (i.e., GCE-PAN@FePc-Ab-BSA immunosensor), using Vibrio cholerae toxin as a case study bioanalyte. Both immunosensors show different charge-transfer kinetics that strongly impact on their immunosensitive detection. From the electrochemical data, GCE-PAN-Ab-BSA is more insulating with the presence of BSA, while the GCE-PAN@ FePc-Ab-BSA is more conducting with BSA. The CV of the GCE-PAN-Ab-BSA is dominated by radial diffusion process, while that of the GCE-PAN@FePc-Ab-BSA is planar diffusion process. The behaviour of GCE-PAN@FePc-Ab-BSA has been associated with the facile coordination of BSA and FePc that permits co-operative charge-transport of the redox probe, while that of the GCE-PAN-Ab-BSA is related to the interaction-induced PAN-BSA insulating state that suppresses charge transport. As a consequence of these different interaction processes, GCE-PAN-Ab-BSA immunosensor provides higher electroanalytical performance for the detection of Vibrio cholerae toxin (with sensitivity of 16.12 Ω/log [VCT, g/mL] and limit of detection (LoD) of 3.20 × 10− 13 g/mL compared to those of the GCE-PAN@FePc-Ab-BSA (4.16 Ω/log (VCT, g mL− 1) and 2.00 × 10− 12 g/mL). The study confirms the need for a thorough understanding of the physico-chemistries of the electrode platforms for the construction of immunosensors. Although this work is on immunosensors for cholera infection, it may well apply to other immunosensors.Item Influence of fermentation on functional properties and bioactivities of different cowpea leaf smoothies during in vitro digestion.(MDPI, 2023-04-19) Moloto, Mapula R.; Akinola, Stephen A.; Seke, Faith; Shoko, Tinotenda; Sultanbawa, Yasmina; Shai, Jerry L.; Remize, Fabienne; Sivakumar, DhariniThis study investigated the effects of Lactiplantibacillus plantarum 75 (LAB 75) fermentation at 37 C for 48 h on the pH, total soluble solids (TSS), colour, total titratable acidity (TTA), carotenoids, and bioactivities of cowpea leaf smoothies from three cultivars (VOP 1, VOP 3, and VOP 4). Fermentation reduced the pH from 6.57 to 5.05 after 48 h. The TTA increased with the fermentation period, whilst the TSS reduced. Fermentation of the smoothies resulted in the least colour changes (DE) in VOP 1 after 48 h. Fermentation of cowpea smoothies (VOP 1, VOP 3, and VOP 4) improved the antioxidant capacity (FRAP, DPPH, and ABTS), which was attributed to the increase in total phenolic compounds and carotenoid constituents in all of the fermented cowpea smoothies. VOP 1 was further selected for analysis due to its high phenolic content and antioxidant activity. The VOP 1 smoothie fermented for 24 h showed the lowest reduction in TPC (11%) and had the highest antioxidant (FRAP, DPPH, and ABTS) activity. Ltp. plantarum 75 was viable and survived the harsh conditions of the gastrointestinal tract, and, hence, could be used as a probiotic. VOP 1 intestinal digesta showed significantly higher glucose uptake relative to the undigested and the gastric digesta, while the gastric phase had higher levels of -amylase and -glucosidase compared to the undigested samples.Item First isolation of glutinol and a bioactive fraction with good anti-inflammatory activity from n-hexane fraction of Peltophorum africanum leaf.(Elsevier B.V., 2016-12-15) Adebayo, Salmon A.; Shai, Leshweni J.; Eloff, Jacobus N.Objective: To investigate the anti-inflammatory activity of different fractions and glutinol (isolated compound), using nitric oxide synthase and cyclooxygenase (COX) inhibition as an indication of anti-inflammatory activity. Methods: Anti-inflammatory activity was evaluated using an in vitro assay determining the inhibition of the activity of pro-inflammatory enzyme model. Cyclooxygenases and inducible nitric oxide synthase are crucial enzymes involved in the pathogenesis of many chronic inflammatory conditions. Results: Sub-fraction F3.3 that was derived from n-hexane fraction of PA leaves significantly inhibited (P = 0.01) the catalytic activity of COX-2 (IC50 = 0.67 mg/mL) better than isolated compound, glutinol (IC50 = 1.22 mg/mL), compound 2 (CP2) (IC50 = 1.71 mg/mL) and sub-fraction F3.3.0 (IC50 = 1.30 mg/mL). A similar trend was observed in investigation of the inhibition of nitric oxide synthesis in RAW 264.7 cells by glutinol, CP2 and F3.3.0. Inducible COX-2 and inducible nitric oxide synthase are among potent signalling enzymes that exacerbate inflammation. Conclusions: Bioactive sub-fractions (F3.3 and F3.3.0) derived from the n-hexane fraction of PA had good anti-inflammatory activity, and the isolated compound, and glutinol may be useful as a template for the development of new anti-inflammatory drugs.Item Annona stenophylla aqueous extract stimulate glucose uptake in established C2Cl2 muscle cell lines.(Academic Journals, 2019-08-21) Taderera, Tafadzwa; Chagonda, Lameck Shoriwa; Gomo, Exnevia; Katerere, David; Shai, Leshweni J.Background: Annona stenophylla is a folk medicine popularly used in Zimbabwe for the treatment of many ailments. This study was carried out to determine some of the possible anti diabetic mechanisms of its action using in vitro cell culturing methods. Methods: A. stenophylla’s effects on glucose uptake were tested using muscle cells (C2Cl2). Expression of glucose 4 transporters was determined by treating cell lines with plant extract. Total RNA was isolated and using RT-PCR, GLUT 4 expression levels were quantified. Translocation of GLUT 4 was assessed using FITC fluorescence measured by flow cytometry. Results: Treatment of cells with plant extract significantly increased glucose uptake in a concentration dependent manner, with the highest concentration (250 µg/ml) giving 28% increased uptake compared to the negative control. The increase in glucose uptake (2.5 times more than control) was coupled to increase in GLUT 4 mRNA and subsequently GLUT 4 translocation. Wortmannin expunged the A. stenophylla induced increase in GLUT 4 mRNA and glucose uptake. Conclusion: The results suggest that A. stenophylla aqueous extract increases glucose uptake partly through increasing the GLUT 4 mRNA and translocation potentially acting via the PI-3-K pathway. This study confirms the ethnopharmacological uses of A. stenophylla indicating potential for anti-diabetic products formulation.Item Investigation of the mechanism of anti- inflammatory action and cytotoxicity of a semipurified fraction and isolated compounds from the leaf of peltophorum africanum (Fabaceae).(SAGE, 2017-05-19) Adebayo, Salmon A.; Steel, Helen C.; Shai, Leshweni J.; Eloff, Jacobus N.Peltophorum africanum extracts have been shown to possess many important medicinal benefits, including anti-inflammatory and antiviral activities. However, the mechanism of action is poorly understood. The mechanism of anti-inflammatory action was determined by measuring the synthesis of cytokines in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophage cells in vitro. Compound 1 (CP1), compound 2 (CP2), and fraction F3.3.0 (F3.3.0) significantly reduced the synthesis of interleukin 1b (IL-1b) from RAW 264.7 cells (1.18, 1.32, and 0.92 ng/mL), respectively. Similarly, CP1, CP2, and F3.3.0 inhibited the production of IL-2 and tumor necrosis factor–a (TNF-a) by RAW 264.7 cells (0.41, 0.60, 0.74 and 0.11, 0.27, 0.24 ng/mL, respectively. In addition, CP1 and CP2 had lower cytotoxicity toward RAW 264.7 cells, with CP2 indicating the lowest cytotoxicity (LD50 ¼ 207.88 mg/mL). The mechanism of action was found to be via the inhibition of pro-inflammation cytokines (IL-1 b and TNF-a). This observation may support the use of P africanum to treat pain-related conditionsItem Natural transmission of foot-and-mouth disease virus between African buffalo (Syncerus caffer) and impala (Aepyceros melampus) in the Kruger National Park, South Africa.(Cambridge University Press, 1999-11-20) Bastos, A.D.S.; Boshoff, C.I.; Keet, D.F.; Bengis, R.G.; Thomson, G.R.VP1 gene sequences of SAT-2 type foot-and-mouth disease (FMD) viruses recovered from impala and African buffalo in the Kruger National Park (KNP) were used to determine intra and interspecies relationships of viruses circulating in these wildlife populations. On this basis five distinct lineages of SAT-2 virus were identified in routine sampling of oesophageo-pharyngeal epithelium from buffalo between 1988 and 1996. Different lineages were associated with discrete geographic sampling localities. Over the period 1985–95, four unrelated epizootics occurred in impala in defined localities within the KNP. Evidence for natural transmission of FMD between buffalo and impala is presented for the most recent 1995 outbreak, with data linking the 1985 and 1988}9 impala epizootics to viruses associated with specific buffalo herds.Item The implications of virus diversity within the SAT 2 serotype for control of foot-and-mouth disease in sub-Saharan Africa.(Microbiology Society Publisher, 2023-02-05) Bastos, A. D. S.; Haydon, D. T.; Sangare, O.; Boshoff, C. I.; Edrich, J. L.; Thomson, G. R.SAT 2 is the serotype most often associated with outbreaks of foot-and-mouth disease (FMD) in livestock in southern and western Africa and is the only SAT type to have been recorded outside the African continent in the last decade. Its epidemiology is complicated by the presence of African buffalo (Syncerus caffer), which play an important role in virus maintenance and transmission. To assess the level of genetic complexity of this serotype among viruses associated with both domestic livestock and wildlife, complete VP1 gene sequences of 53 viruses from 17 countries and three different host species were analysed. Phylogenetic analysis revealed eleven virus lineages, differing from each other by at least 20% in pairwise nucleotide comparisons, four of which fall within the southern African region, two in West Africa and the remaining five in central and East Africa. No evidence of recombination between these lineages was detected, and thus we conclude that these are independently evolving virus lineages which occur primarily in discrete geographical localities in accordance with the FMD virus topotype concept. Applied to the whole phylogeny, rates of nucleotide substitution are significantly different between topotypes, but most individual topotypes evolve in accordance with a molecular clock at an average rate of approximately 0?002 substitutions per site per year. This study provides an indication of the intratypic complexity of the SAT 2 serotype at the continental level and emphasizes the value of molecular characterization of diverse FMD field strains for tracing the origin of outbreaks.Item Characterisation of a SAT-1 outbreak of foot-and-mouth disease in captive African buffalo (Syncerus caffer): Clinical symptoms, genetic characterisation and phylogenetic comparison of outbreak isolates.(Elservier, 2006-11-02) Vosloo, W.; De Klerk, L.-M.; Boshoff, C.I.; Botha, B.; Dwarka, R.M.; Keet, D.; Haydon, D.T.African buffalo (Syncerus caffer) play an important role in the maintenance of the SAT types of foot-and-mouth disease (FMD) in southern Africa. These long-term carriers mostly become sub-clinically infected, maintaining the disease and posing a threat to other susceptible wildlife and domestic species. During an unrelated bovine tuberculosis experiment using captive buffalo in the Kruger National Park (KNP), an outbreak of SAT-1 occurred and was further investigated. The clinical signs were recorded and all animals demonstrated significant weight loss and lymphopenia that lasted 100 days. In addition, the mean cell volume and mean cell haemoglobin values were significantly higher than before the outbreak started. Virus was isolated from several buffalo over a period of 167 days post infection and the molecular clock estimated to be 3 x 10-5 nucleotide substitutions per site per day. Seven amino acid changes occurred of which four occurred in hypervariable regions previously described for SAT-1. The genetic relationship of the outbreak virus was compared to buffalo viruses previously obtained from the KNP but the phylogeny was largely unresolved, therefore the relationship of this outbreak strain to others isolated from the KNP remains unclear.Item Genetic characterisation of African swine fever viruses from outbreaks in southern Africa (1973–1999).(Elsevier, 2006-11-15) Boshoff, C.I.; Bastos, A.D.S.; Gerber, L.J.; Vosloo, W.African swine fever (ASF) is a highly lethal and economically significant disease of domestic pigs in the southern African sub-region, where outbreaks regularly occur. There is anecdotal evidence suggesting that trans-boundary movement of infected animals may have played a role in precipitating widespread outbreaks in the past, however, since the 1970s outbreaks have generally been more localised, particularly in those countries where control of animal movement is strictly regulated. The origin and relatedness of regional ASF outbreaks was investigated here by means of a two-step genetic characterisation approach whereby p72 gene sequencing was used to delineate genotypes, prior to intra-genotypic resolution of viral relationships by central variable region (CVR) characterisation of the 9RL ORF. In this manner, regional virus heterogeneity and epidemiological links between outbreaks could be assessed for the first time through phylogenetic analysis of the C-terminal end of the p72 gene of viruses recovered from domestic pig outbreaks in southern Africa between 1973 and 1999. The phylogeny revealed the presence of 14 distinct p72 genotypes of which 6 (genotypes XVII–XXII) were considered novel. Eight of these were country specific with the remaining six having a trans-boundary distribution. CVR products were heterogeneous in size ranging from 377 bp to 533 bp across the 14 southern African genotypes. Within-genotype CVR comparisons revealed the presence of a genotype XIX virus with an extended field presence in South Africa (1985–1996) and permitted discrimination between three genotype VII viruses that were identical across the p72 gene.Item First molecular assessment of the African swine fever virus status of Ornithodoros ticks from Swaziland.(BMC Commercial Publisher, 2014-10-03) Boshoff, Carin I.; Bastos, Armanda D.S.; Dube, Mzwandi M.; Heath, LivioAfrican swine fever (ASF) is an economically significant haemorrhagic disease of domestic pigs. It is caused by the African swine fever virus (ASFV), a deoxyribonucleic acid (DNA) arbovirus. Argasid ticks of the genus Ornithodoros, which are widely distributed throughout southern Africa, play a primary role in virus maintenance and spread within the endemic sylvatic cycle. The ASF status of Swaziland is unknown, but this land-locked country is surrounded by ASF-positive countries, has a burgeoning pig industry and sylvatic cycle hosts present within its borders. In this first assessment of ASF status, warthog burrows in seven nature reserves and game management areas in Swaziland were investigated for tick and virus presence. Tick infestation rates of between 33.3% – 88.8% were recovered for the four Ornithodoros-infested reserves. A total of 562 ticks were screened for virus genome presence using a duplex Polymerase Chain Reaction (PCR) that targets the C-terminal end of the p72 gene of the ASFV and confirms DNA integrity through amplification of the 16S rRNA tick host gene. All samples were negative for virus genome presence and positive for the tick genome target. Nucleotide sequencing of the latter confirmed that Ornithodoros ticks from Swaziland are identical to those from the Kruger National Park in South Africa across the gene region characterised. Whilst this first evaluation of ASF presence in Swaziland indicates that the virus does not appear to be present in the key virus vector, the presence of sylvatic cycle hosts, together with the country’s proximity to ASF-affected countries calls for expanded investigations and regular monitoring of the ASF status of Swaziland.Item Genome sequences of three African swine fever viruses of genotypes I, III, and XXII from South Africa and Zambia, isolated from ornithodoros soft ticks.(Microbiology Society, 2020-03-05) Ndlovu, S.; Williamson, A.L.; Malesa, R.; Van Heerden, J.; Boshoff, C.I.; Bastos, A.D.S; Heath, L.; Carulei, O.Here, we report the draft genome sequences of three African swine fever viruses isolated from Ornithodoros soft ticks. Isolates LIV 5/40 (Zambia), SPEC 57 (South Africa), and RSA/2/2008 (South Africa) belong to genotypes I, III, and XXII, respectively.Item Non-enzymatic glycation and formation of advanced glycation end-products alters the activity and related kinetic properties of aldose reductase.(Wiley, 2021-07-26) Mkhombo, M.H.; Mogale, M.A.; Shai, L.J.; Lebelo, S.L.Aldose reductase was incubated with and without either fructose or glucose for 42 days to initiate the glycation process. The concentrations of fructosamine were measured on every 7th day using the standard nitroblue tetrazolium reagent assay. Carboxymethyllysine formed was determined using enzyme linked immunosorbent assay-based methods, fluorescent end-products were measured using spectrofluorometric methods. Activities were assayed by measuring the absorbance of co-factor nicotinamide adenine dinucleotide phosphate hydrogen at 340 nm. Fructosamine, carboxymethyllysine protein adducts and fluorescent end-products were significantly higher (p < 0.001) when aldose reductase was incubated with fructose or glucose than without. Although the glycation of aldose reductase did not result in the alteration of both the optimum pH and temperature of the enzyme, both the activity and Vmax were increased, whereas Km was decreased. Nonenzymatic glycation of aldose reductase increases both its activity and Vmax, while decreasing its Km. Additionally, glycation did not affect the pH of enzyme and temperature optima.Item A preliminary investigation of exposure to rabies virus in selected wildlife in the Kruger National Park, South Africa.(AOSIS, 2021-03-14) Rossouw, Leana; Boshoff, Carin; Sabeta, Claude; Kotzé, JohannRabies is a zoonotic disease caused by members of the genus Lyssavirus and causes fatal encephalitis in warm-blooded vertebrates. Rabies has been previously confirmed in domestic dog populations in close proximity to the Kruger National Park (KNP) and can potentially threaten conservation efforts. Domestic dogs infected with rabies virus occasionally enter the KNP and may be a source of rabies exposure to wildlife. Therefore, the aim of this study was to determine if wild carnivores in the KNP have been exposed to rabies virus, based on the presence of antibodies. Serum samples from the African wild dog (Lycaon pictus), spotted hyena (Crocuta crocuta), lion (Panthera leo), leopard (Panthera pardus) and banded mongoose (Mungos mungo) were tested for the presence of rabies-specific antibodies using the BioPro enzyme-linked immunoassay kit (BioPro ELISA kit). Selected sera were tested in parallel with the fluorescent antibody virus neutralisation test (FAVNT). Of the 168 carnivore serum samples screened, eight (4.8%) had a percentage blocking (PB) ≥ 40, indicating the presence of rabies-binding antibodies and confirmed with the FAVNT to be very low levels of rabies virus neutralising antibodies (range 0.00 IU/mL – 0.22 IU/mL). Rabies-binding antibodies detected by the BioPro ELISA kit and rabies virus neutralising antibodies shown by the FAVNT should however be interpreted with caution because of the lack of validation and species-specific cutoff values for wild carnivores. Conservation implications: The results of this study will assist in understanding the epidemiology of rabies in the KNP carnivores, especially exposure risk. The use of rabies diagnostic tools developed for domestic animals for disease surveillance in the KNP carnivores was also evaluated and the outcomes will further support research on rabies in free-ranging wildlife populations.Item Experimental infection of giraffe (Giraffa camelopardalis) with SAT-1 and SAT-2 foot-and-mouth disease virus.(Wiley, 2010-11-09) Vosloo, W.; Swanepoel, S.P.; Bauman, M.; Botha, B.; Esterhuysen, J.J.; Boshoff, C.I.; Keet, D.F.; Dekker, A.The potential role of giraffe (Giraffa camelopardalis) in the epidemiology and spread of foot-and-mouth disease (FMD) SAT types was investigated by experimental infection and detection of virus in excretions using virus isolation on primary pig kidney cell cultures. In two experiments separated by a period of 24 months, groups of four animals were needle infected with a SAT-1 or SAT- 2 virus, respectively and two in-contact controls were kept with each group. Viraemia was detected 3–9 days post-infection and virus isolated from mouth washes and faeces only occasionally up to day 13. The SAT-1 virus was transmitted to only one in-contact control animal, probably via saliva that contained virus from vesicles in the mouth of a needle-infected animal. None of the animals infected with the SAT-2 virus had any vesicles in the mouth, and there was no evidence of transmission to the in-contact controls. No virus was detected in probang samples for the duration of the experiments (60 days postinfection), indicating that persistent infection probably did not establish with either of these isolates. Giraffe most likely do not play an important role in FMD dissemination. Transmission of infection would possibly occur only during close contact with other animals when mouth vesicles are evident.Item Cassia abbreviata enhances glucose uptake and glucose transporter 4 translocation in C2C12 mouse skeletal muscle cells.(SAGE, 2021-01-21) Kamga-Simo-III, F. D. Y.; Kamatou, G.P.; Ssemakalu, C.; Shai, L.J.Background. This study aims at assessing C. abbreviata aqueous extracts for its potential to exhibit anti-diabetic activity in skeletal muscle cells. In addition to the toxicological and glucose absorption studies, the action of C. abbreviata extracts on some major genes involved in the insulin signaling pathway was established. Methods. The in vitro cytotoxic effects C. abbreviata was evaluated on muscle cells using the MTT assay and the in vitro glucose uptake assay conducted using a modified glucose oxidase method described by Van de Venter et al. (2008). The amount of GLUT-4 on cell surfaces was estimated quantitatively using the flow cytometry technique. Real time quantitative PCR (RT-qPCR) was used to determine the expression of GLUT-4, IRS-1, PI3 K, Akt1, Akt2, PPAR-g. Results. Cytotoxicity tests revealed that all extracts tested at various concentrations were nontoxic (LC50 > 5000). Aqueous extracts of leaves, bark and seeds resulted in a dose-dependent increase in glucose absorption by cells, after 1 h, 3 h and 6 h incubation period. Extracts of all three plant parts had the best effect after 3 h incubation, with the leaf extract showing the best activity across time (Glucose uptake of 29%, 56% and 42% higher than untreated control cells after treatment with 1 mg/ml extract at 1 h, 3 h and 6 h, respectively). All extracts, with the exception 500 mg/ml seed extract, induced a two-fold increase in GLUT-4 translocation while marginally inducing GLUT-10 translocation in the muscle cells. The indirect immunofluorescence confirmed that GLUT-4 translocation indeed occurred. There was an increased expression of GLUT-4, IRS1 and PI3 K in cells treated with insulin and bark extract as determined by the RT-qPCR. Conclusion. The study reveals that glucose uptake involves GLUT-4 translocation through a mechanism that is likely to involve the upstream effectors of the PI3-K/Akt pathway.