Theses and Dissertations (Paraclinical Sciences)

Permanent URI for this collectionhttp://hdl.handle.net/2263/32298

Browse

Now showing 1 - 20 of 167

Efficacy of selected southern African plants used in ethnoveterinary medicine against ticks and associated infections
(University of Pretoria, 2023-07) McGaw, Lyndy Joy; Makhubu, Fikile; Steyl, Johan Christian Abraham; tamspowell@gmail.com; Powell, Tamarin
Ticks cause many problems in cattle worldwide, including transmission of tick-borne diseases and quality issues. Attachment of ticks to host organisms causes wounds which enhance the likelihood of secondary infections and abscesses. The aim of this project was to select southern African plants used in ethnoveterinary medicine to repel or kill ticks, to confirm their efficacy against ticks, and to investigate the activity of the plant extracts against bacteria and fungi implicated in causing secondary infections accompanying tick infestations. Eight plants were selected and extracted using acetone, ethanol and sterile distilled water. The extracts were tested against tick larvae using repellence and mortality assays. The minimum inhibitory concentration of each extract against bacterial strains of Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive), as well as the yeast fungal strain Candida albicans, was determined using the broth microdilution assay. Extracts were also tested for their ability to prevent and disrupt bacterial biofilm formation, and for cytotoxicity against bovine dermis and Vero cells. In general, the extracts had better tick repellence than acaricidal effects. Aloe ferox extracts had the highest repellence against tick larvae, as well as the best acaricidal effects at a concentration of 10 mg/ml. Ethanolic extracts of Aloe ferox (MIC = 0.05 mg/ml) and Lavandula lanata (MIC = 1.25 mg/ml) had the best antibacterial activity against E. coli and S. aureus respectively, whereas the acetone extract of Ptaeroxylon obliquum (MIC = 0.02 mg/ml) had the best antifungal activity. For the biofilm inhibitory activity, water extracts of A. ferox (92%), P. obliquum (91%) and Tulbaghia violacea (97%) were able to strongly inhibit biofilm formation after 24 h of treatment. Overall, T. violacea showed promising results with percentage inhibitions close to 100% for all three extracts after 24 h of extract treatment against biofilm formation. After 48 h, only the water extracts continued to inhibit the biofilm effectively. Aloe ferox had SI values as high as 4.88 and 18.75 against Vero cells and BD cells respectively. In summary, water extracts had strong antibiofilm and tick repellence activity, but were not acaricidal, antibacterial or antifungal. Ethanolic extracts had good overall activity in all the assays, and Aloe ferox and Lippia javanica were the most active plant species in the study.
Induction of lysogenic bacteriophages of Shiga toxin-producing Escherichia coli by antimicrobial growth promoters used in food- producing animals in South Africa
(University of Pretoria, 2023) Karama, Musafiri; Marufu, Munyaradzi Christopher; nomondengoma96@gmail.com; Ngoma, Nomonde N.F.
Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a foodborne disease characterized by diarrhoea and complications such as haemorrhagic colitis and the haemolytic uremic syndrome, a complication which can lead to kidney failure in 5-10% of humans showing STEC disease. The major virulence factors of STEC are two toxins (Stx1 and Stx2) encoded on bacteriophages, commonly termed Shiga toxin-converting bacteriophages (stx-converting bacteriophages). Previous studies have shown that a number of antimicrobials which are used for livestock growth promotion can induce stx-converting bacteriophages at subinhibitory concentrations. Induced stx-converting bacteriophages are considered the main “drivers” of STEC emergence and evolution as they transfer bacteriophage-encoded Shiga toxin-encoding genes from STEC to naïve E. coli by transduction. This phenomenon is considered to be behind the formation of novel STEC strains. Although a European Union-wide ban on the use of antimicrobial growth promoters in animal agriculture exists since 2006, this controversial practice remains in effect in a number of countries around the world including South Africa. Therefore, in this study, four antimicrobials which are approved for livestock growth-promotion in South Africa were tested at sub-inhibitory concentrations for their capacity to induce Shiga toxin-converting bacteriophages from 47 STEC O157:H7 isolates using the double-layer agar-plaque assay. The antimicrobials tested included josamycin, virginiamycin, flavophospholipol and poly 2-propenal 2-propenoic acid. These antimicrobial growth promoters had never been tested for their capacity to induce bacteriophages. In addition, induced bacteriophages were characterized for the presence of genes encoding different Shiga toxin subtypes (stx1, stx2, stx2c and stx2d), restriction fragment length polymorphisms and morphology by electron microscopy. The following bacteriophage induction rates were obtained for each antimicrobial growth promoter tested: poly 2-propenal 2-propenoic acid, 42.6% (20/47); virginiamycin, 34.0% (16/47); josamycin, 34.0% (16/47); flavophospholipol, 29.8% (14/47). A small number of STEC O157:H7 isolates induced bacteriophages spontaneously (14.9%; 7/47). Most of the induced bacteriophages carried the stx2 and stx2c-encoding genes, independent of the induction method while only a few bacteriophages carried stx2d except for josamycin and spontaneously-induced bacteriophages which stx2d at higher rates of 87.5% (14/16) and 100% (7/7). Electron microscopy revealed only four representative groups of virion particle morphologies: three groups of bacteriophages with either a long hexagonal, oval/circular, and elongated head which all had long tails and one group of bacteriophages with an icosahedral/hexagonal head and a thick contractile tail. These results showed that josamycin, virginiamycin, flavophospholipol and poly 2-propenal 2-propenoic acid induce stx-converting bacteriophages from STEC O157:H7. Induced bacteriophages were largely stx2 and stx2c positive, but stx2d positive to a lesser extent. Induction of stx-converting bacteriophages by antimicrobial growth promoters may be contributing to the conversion of naïve E. coli into virulent STEC strains as a result of bacteriophage transduction and horizontal transfer of stx-encoding genes from STEC to naive E. coli. This phenomenon has been identified as the driving force behind STEC emergence, expansion and evolution. The formulation of policies and implementation of strategies which promote the prudent use of antimicrobials growth promoters in animal agriculture in South Africa and elsewhere where these compounds are still in use are recommended.
Seasonal physiological responses in the greater thick-tailed galago (Otolemur crassicaudatus)
(University of Pretoria, 2023) Tordiffe, Adrian Stephen Wolferstan; Scheun, Juan; channen1221@gmail.com; Long, Channen
Primate populations over the globe are facing declines as a result of several factors including climate change. It has become imperative to gain further insight into how primate species respond to these changes in weather to ensure appropriate conservation approaches. For this study, I chose to monitor the physiological changes of a population of greater thick-tailed galagos (O. crassicaudatus) residing in a highly seasonal, temperate environment. Research of this strepsirrhine species has been lacking for over two decades and the scientific community is unaware how they respond to seasonal weather changes. In this study, we assessed their glucocorticoid and thyroid hormone levels to monitor their hormonal responses, gut microflora, and metabolite profiles associated with changes in temperature and food availability. We successfully validated the immunoassays necessary to measure hormone metabolites in this species. The results revealed an increase in hormone levels during the summer season which may be caused by an increase in energy expenditure as food availability and temperatures increase. Furthermore, lactating females during this time require additional energy and nutrition to sustain themselves and their offspring. The results of the metabolite analyses indicate these concentrations were affected by changes in diet. However, it appears the dominant microflora and metabolic pathways adapt to seasonal fluctuations of nutrient intake to ensure the body receives the essential amino acids needed for ATP generation. Overall, this project has given further information into the mechanisms undertaken by this species during times of low food availability and will assist in future primate conservation.
Pharmacological activity of South African medicinal plants and mechanism of action against Staphylococcus aureus isolated from clinical bovine mastitis
(University of Pretoria, 2023-11) McGaw, Lyndy Joy; Petzer, Inge-Marie; Dzoyem, Jean Paul; chineloerhabor@gmail.com; Erhabor, Rosemary Chinelo
Bovine mastitis is an inflammation of the mammary parenchyma which is usually caused by an infection that could stem from microorganisms in an already infected udder of another cow, or from the environment. Bovine mastitis infection is mostly caused by Staphylococcus and Streptococcus species and has gained global importance due to the increase of multi-drug resistant bacteria, and resistance to common antibiotic therapy. Most staphylococci associated with bovine mastitis can express biofilm which can prevent and reduce the effects of antibacterial agents and the efficacy of the leucocytes. Antimicrobial resistance is a naturally occurring process that can be accelerated with the overuse or incorrect use of antibiotics. The World Health Organization has declared antimicrobial resistance as one of the top 10 most important public health threats. Antimicrobial resistance reduces the efficacy of treatment which may lead to an increase of chronic udder parenchyma infections/diseases with no cure, causing in some cases organ failure and death. Some mastitis pathogens developed resistance to antimicrobials due to their ability to form biofilm and spread this characteristic through the processes of transformation, conjugation and transduction. Gene replication during biofilm formation can lead to mutation (remodeling of the gene), and bacteria during this process protect themselves by producing extracellular polymeric substances leading to antibiotic resistance. Following a comprehensive literature survey, twelve medicinal plants growing in South Africa (Combretum molle, C. erythrophyllum, C. caffrum, C. elaeagnoides, C. padoides, Tithonia rotundifolia, Leonotis leonurus, L. nepetifolia, Rosmarinus officinalis, Bauhinia tomentosa, Maytenus peduncularis and Faurea saligna) used traditionally for the treatment of inflammation, fever, breast swelling, pain and wounds were selected in this study. The dried plant leaves were separately extracted with six different solvents, namely methanol, ethanol, acetone, dichloromethane: methanol (ratio 1:1), and cold and hot water following standard procedures. Antibacterial assays were conducted, including determination of the minimum inhibitory concentration (MIC), antibiofilm, and anti-quorum sensing activity. The antibacterial activity was investigated against six clinical isolates of Staphylococcus aureus from bovine mastitis cases (Ethics permission number V121_ 16, University of Pretoria) and two Staphylococcus ATCC strains (S. aureus ATCC 29213, S. epidermidis ATCC 35984) which were collected from the Milk Laboratory, Department of Production Animal Studies, Faculty of Veterinary Sciences, University of Pretoria, and Chromobacterium violaceum ATCC 12472 (available in the phytomedicine program bio-bank for the anti-quorum sensing activity. The MIC assay was carried out using the serial microdilution method. Antibiofilm activity of the extracts was determined at three different times, time zero (T0) which is at the planktonic stage of the bacterial cells for antiadhesion activity, time 24 (T24), which was done after 24 h of incubation to allow cell attachment and biofilm formation and at time 48 (T48) when the biofilm was at its full-grown matured stage using two different assays. The crystal violet assay was used to determine the anti-adhesion and reduction/inhibition of biofilm biomass production while the metabolic p-iodonitrotetrazolium violet (INT) assay was used to determine the bactericidal effect of the extracts on biofilm metabolic/respiratory activity. The antioxidant activity and anti-inflammatory effects of the extracts were determined using two antioxidant and inflammatory assays. For antioxidant activity the in vitro DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ABTS (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) electron reduction assays were used, and the 15-lipoxygenase enzyme (15-LOX) and nitric oxide (NO) inhibitory assays were employed to determine the anti-inflammatory activity. The cytotoxicity of the extracts was determined against bovine dermis and Vero cells using the 3-(4, 5-dimethylthiazolyl-2)-2.5-diphenyltetrazolium bromide (MTT) colorimetric assay. Finally, Synergistic screening of the extracts with the best antibacterial activity against the pathogens were tested in combination with different antibiotics using the checkboard method. From this study, of the six extractant, the acetone extract had good antibacterial activity against five of the pathogens compared with other extractants. The extracts had outstanding to weak MIC values ranging between 0.02 and 2.5 mg/mL. The extracts showed commendable inhibition against cell attachment and biofilm formation in the test organisms and inhibited the metabolic activity of the pathogens (bactericidal effect). The extracts at different concentrations exhibited good quorum quenching potential and had the ability to inhibit the production of nitric oxide and inhibited the 15-lipoxygenase enzyme. Most of the extracts were safe to both the Vero and bovine dermis cells. The Combretum padoides extracts (except the water extracts against some of the pathogens) had outstanding broad-spectrum antibacterial activity against all test organisms with MIC values of 0.02 – 0.16 mg/mL. Rosmarinus officinalis extracts also had good MIC values against the test organisms (MIC = 0.02 - 1.67 mg/ml). All extracts had varying antiadhesion and biofilm inhibition activity against the test organisms at different test times and showed bactericidal effects to the bacteria. The extracts of Leonotis leonorus had the best broad-spectrum antibiofilm activity (anti-adhesion and inhibition of the metabolic activity) at the three different test times with inhibition >50%. Combretum padoides inhibited the production of biofilm biomass in at least five of the test organisms and inhibited metabolic activity by more than 50% in six of the tested strains. Faurea saligna also inhibited the metabolic activity in at least seven (7) of the tested bacterial strains at the different test times. All extracts had violacein inhibitory activity, but the Combretum species as well as R. officinalis and F. saligna extracts had the best violacein inhibition while the extracts of C. padoides had outstanding anti-quorum sensing activity with violacein inhibition at all concentrations (0.08 to 2.5 mg/mL). The extracts of C. molle, C. padoides, F. saligna, R. officinalis, M. peduncularis, C. caffrum, and the methanol extract and fractions of C. elaeagnoides had good bactericidal effects against C. violaceum with the minimum bactericidal concentration (MBC) at 0.31 to 1.25 mg/mL respectively. This result suggests the anti-quorum sensing ability of the extracts, which have potential for further study in the management of disease virulence factors and may prevent the multiplication (biofilm formation) in organisms causing microbial infections. All extracts showed excellent to weak antioxidant potential using the DPPH and ABTS methods. Six of the extracts had the best DPPH scavenging activity (EC50 0.67 - 1.90 μg/mL respectively) when compared to the ascorbic acid and Trolox (EC50 7.11 and 6.20 μg/mL).
In vitro studies of Searsia lancea leaf extracts against multi-drug resistant bacterial isolates from clinical cases of bovine mastitis
(University of Pretoria, 2023) McGaw, Lyndy Joy; Petzer, Inge-Marie; thelchema@gmail.com; Akinboye, Ayodele Omolade
Introduction Bovine mastitis, a common inflammatory disease affecting mammary glands of dairy cattle, has severe implications for milk quality and production. It is a critical pathology in global dairy herds, and poses a significant economic burden on the dairy industry, leading to substantial financial losses due to decreased production and increased culling. Bovine mastitis is a significant problem in the dairy industry due to antibiotic-resistant pathogens forming biofilms via quorum sensing. Pathogenic microorganisms, particularly Streptococcus species, Staphylococcus species and Escherichia (E.) coli, are the major aetiological agents. Managing mastitis typically involves antibiotics, but the continuous use of conventional antimicrobial agents has led to antimicrobial resistance and treatment failures. The ability of bacteria to form biofilms is associated with resistance, while quorum sensing plays a vital role in biofilm formation. This complex interplay between quorum sensing (QS) and biofilm formation challenges mastitis management, necessitating the development of alternative therapeutics to combat this microbial threat effectively. In addition, managing inflammation with its accompanying oxidative stress in mastitis is a holistic approach in managing the disease. Plants have been shown to demonstrate antioxidant and anti-inflammatory activities, and show promise in restoring drug sensitivity and enhancing host immunity, therefore, to address this issue, researchers are exploring plant-derived antimicrobial agents. This study, therefore, aimed to investigate the antibiotic resistance profile of major bacterial isolates from mastitis cases, and to determine the antibacterial, antibiofilm and anti-quorum sensing, antioxidant and anti-inflammatory activities and cytotoxicity of the extracts and fractions of selected South African plants. This will serve to contribute to the development of alternative treatments for bovine mastitis caused by resistant bacteria. The selection of plants (Searsia lancea, Erythrina caffra, Antidesma venosum, and Indigofera frutescens) was based on reported minimum inhibitory concentration (MIC) of less than 0.1 mg/mL against staphylococcal bacteria of mastitis origin (Akinboye et al., 2023). The antimicrobial susceptibility of the bacterial strains was determined using a disc diffusion method, while the biofilm forming ability (BFA) of bacterial isolates in Brain Heart Infusion broth (BHI) and Tryptic Soy broth (TSB) was determined. Furthermore, the antibacterial, antibiofilm and anti-quorum sensing activities of the extracts and fractions were determined against a panel of mastitis pathogens using a two-fold serial microdilution assay. In addition, v their antioxidant and anti-inflammatory activities as well as their effects on mRNA expression of their pro-inflammation cytokine genes were investigated using standard methods, while their cytotoxicity was determined against Vero cells and bovine dermis cells. Among 32 clinical bacterial isolates tested, 84.38% (27) exhibited antibiotic resistance, with 55.56% (15) being multidrug-resistant. These include 100% (7) of the E. coli isolates, 50% (2) of the staphylococcal isolates, 42.86% (3) of Streptococcus (Str.) uberis, and 14.29% (1) each of Str. agalactiae and Str. dysgalactiae strains. These results reveal a concerning trend of high antibiotic resistance among clinical isolates, with a significant portion being multidrug- resistant. Remarkably, E. coli isolates showing 100% resistance, along with varying resistance in other strains, pose challenges for conventional antibiotic effectiveness and highlight the necessity for targeted strategies due to specific strain resistances, especially in cases of E. coli- associated mastitis in dairy cattle. Moreover, all plant extracts exhibited antibacterial activity against all the bacterial isolates, but S. lancea demonstrated better activity compared to other plants. The MIC values obtained ranged between 0.01 - 2.50 mg/mL, with the narrowest range obtained with the acetone extract of S. lancea (MIC = 0.01 – 0.57 mg/mL). The overall MIC range for all the extracts against the E. coli isolates was 0.01 – 0.31 mg/mL, which showed that the extracts exhibited good to moderate activities against these multidrug resistant (MDR) E. coli isolates. The lowest MIC observed (0.01 ± 0.00 mg/mL) was demonstrated by acetone and ethanol extracts of S. lancea. Interestingly, the acetone extract of S. lancea also showed good activity against 80% (24) of the 30 organisms tested, followed by the ethanol extract (60%). Though, the acetone extract of S. lancea demonstrated the highest total antibacterial activity (TAA) against E. coli, the ethanol extract of S. lancea demonstrated higher efficacy (TAA) against three of the four species of isolates. Also, when considering individual isolates, the ethanol extract of S. lancea exhibited an exceptional TAA value of 15 790 mL/g against ECO4, and TAA values above 1 000 mL/g against 83.33% (25 out of 30) of the organisms which is more than 56.67% (17) by its acetone extracts. This suggests the ethanol extract is more efficacious than the acetone extract. The dichloromethane (DCM) and ethyl acetate fractions of ethanol extracts of S. lancea had good antibacterial activity against the isolates with MIC values as low as 0.001 and 0.01 mg/mL, respectively. In comparison to the reference antibiotic, ciprofloxacin, the DCM and ethyl acetate fractions demonstrated better antibacterial activity, highlighting the potential of these fractions as alternatives to commonly used antibiotics against mastitic pathogens. Interestingly, the crude ethanol extract demonstrated better antibacterial activity against Str. agalactiae, Str. vi uberis, E. coli and Sta. aureus isolates than all the fractions, while in sharp contrast, all the fractions demonstrated better activities than the extract against Str. dysgalactiae isolates. This suggests species-specific additional, synergistic or antagonistic antibacterial activities between the compounds in the extract, and this infers fractionation of the extracts potentiates or attenuates the effects of the compounds in the extract against the bacteria. Moreover, the extracts, particularly from S. lancea, demonstrated low cytotoxicity toward BD and Vero cells, crucial for ensuring safety in potential therapeutic applications. Remarkably, S. lancea displayed the highest mean selectivity index value of 25.70, indicating its selectivity in targeting bacterial pathogens while being less harmful to host cells. The fractions exhibited minimal cytotoxic effects on bovine dermis (BD) cells. The selectivity index values of S. lancea extracts and DCM fraction were remarkably high, reaching 100 and 90, respectively, further emphasizing their safety and selectivity in targeting bacteria while being non-toxic to mammalian host cells. Out of the 29 isolates tested, 93.10% (27) and 68.97% (20) demonstrated BFA in TSB and BHI, respectively. Compared to BHI, TSB appeared to enhance BFA of the bacteria except for Str. uberis strains. These findings highlight a significant prevalence of BFA among isolates, emphasizing potential biofilm-related challenges in bovine mastitis management. Moreover, the varying BFA between different growth media suggests a crucial influence of the medium on biofilm formation. Additionally, the strain-specific behaviour, notably seen in Str. uberis strains, underscores the need for nuanced, tailored approaches when managing different bacterial strains causing mastitis. Furthermore, biofilm inhibition and eradication tests revealed varying activities of extracts and fractions, with S. lancea displaying the most potent antibiofilm activity at varying sub-MIC concentrations. All the plants demonstrated good biofilm disruption ability (BDA) against 24 h preformed biofilms of the isolates except for E. caffra, while S. lancea displayed good BDA against all the 48 h preformed biofilms of the bacteria. Generally, the plants' antibiofilm activities appeared to improve as the biofilm matured, with few exceptions. In addition, DCM and ethyl acetate fractions generally exhibited good inhibition at various concentrations against most of the bacterial strains tested, but the ethyl acetate fraction had the best antibiofilm and preformed biofilm disrupting activities. The differing abilities of extracts and fractions to inhibit and eradicate biofilms highlight the complexity of managing biofilm-related infections in mastitis. Searsia lancea stands out with potent antibiofilm activity at sub-MIC vii concentrations, suggesting its potential as a key player in combating biofilm-forming bacteria associated with mastitis. While most plants demonstrated good BDA against 24 h preformed biofilms, S. lancea displayed consistent effectiveness against even longer matured 48 h biofilms of bacteria. This suggests a promising ability to disrupt established biofilms, a crucial aspect in managing chronic or recurrent mastitis cases. The ethanol extracts of S. lancea demonstrated a minimum quorum sensing inhibition concentration (MQSIC) at 2.50 mg/mL, and the lowest MQSIC50 value (< 0.08 mg/mL), demonstrating its quorum quenching ability. This indicates its potential to interfere with bacterial communication systems, disrupting their ability to coordinate and cause infections, which could be pivotal in controlling mastitis. Remarkably, the DCM and water fractions demonstrated the lowest MQSIC value, with DCM having the lowest MQSIC50 value of 0.01 mg/mL, which suggests that the fractions have the potential to modulate virulence factors by specifically inhibiting the formation of the violacein pigment indicating inhibition of quorum sensing at sub-MIC levels. Lastly, S. lancea demonstrated better antioxidant and anti-inflammatory activities compared to other extracts, and was able to downregulate the expression of iNOS gene by 99% while upregulating the expression of COX-2 and IL-6 in LPS-stimulated RAW 264.7 macrophages. This indicates its potential to tackle both oxidative stress and inflammatory expression of iNOS as well as enhance immune response in immunocompromised cows. In conclusion, this research highlights the urgency of tackling AMR in livestock management, and demonstrates the promising therapeutic potential of these plants, particularly S. lancea, in treating bovine mastitis. The study also showed that the extracts exhibited significant antibacterial activity against these drug-resistant strains, as S. lancea had the most potent antibacterial activity and appeared promising as a broad-spectrum antibacterial agent against mastitis-related bacteria. Importantly, it had a moderate cytotoxicity profile, particularly the ethanol extracts and its fractions. This suggests that S. lancea extracts could serve as a valuable resource for developing novel treatments. Moreover, this study underscores the potential of fractions of the S. lancea extract as valuable resources in combating antimicrobial resistant mastitis pathogens. Further investigation of their mechanisms, identification of active compounds, and thorough in vivo assessments are essential steps toward their development as effective preventative and treatment options for microbial infections such as bovine mastitis. Furthermore, the ability of these plants, especially S. lancea, to inhibit QS and biofilms at various developmental stages may play a pivotal role in managing mastitis infections and curbing the emergence of antimicrobial resistance. Nonetheless, further research is needed to elucidate the precise mechanisms underlying the inhibition of quorum signalling and biofilms, and to identify the specific compounds responsible for the observed activities. Overall, the study highlights S. lancea as a promising candidate alternative for treatment of infection, oxidative stress and inflammation in bovine mastitis, underlining the importance of further research on the plant and the active compounds within its fractions, with the intent of developing S. lancea into a global South African product in the management of bovine mastitis worldwide.
Antimicrobial and other beneficial properties of fodder species with potential use in controlling diarrhoeagenic Escherichia coli in cattle
(University of Pretoria, 2023) McGaw, Lyndy Joy; Famuyide, Ibukun Michael; Elgorashi, Esam E.; Kgosana, Gloria; moipone25@yahoo.com; Lebeloane, Moipone Mary
Shiga toxin-producing E. coli (STEC) is a diarrhoeagenic pathogen commonly transmitted from cattle to other animals via contaminated meat, milk and environmental water sources, causing a serious public health burden with major economic implications in livestock and human health. Diarrhoeagenic E. coli harbour biofilm-forming and quorum sensing characteristics involved in multidrug resistance (MDR). Multidrug resistance is challenging in combating infectious veterinary and zoonotic diseases, prompting restrictions on prophylactic use of antimicrobials as feed additives, especially for growth promotion in young animals. In addition, microbial infections also contribute to inflammatory and oxidative disorders in immune-compromised young or old animals, negatively affecting production and health. Thus, there is an urgent need to discover novel alternative or complementary feed additives with nutritional value, antimicrobial, antibiofilm, and anti-inflammatory properties to maintain cattle gastrointestinal health, increase growth rate, and maximise production efficiency. In African countries including those in southern Africa, there is substantial application of various plant species in cattle production for use as alternative animal feed or as anti-diarrhoeal remedies against infectious pathogens. Many of the same plant species have been traditionally used to treat various diarrhoeal and inflammatory conditions in humans. Hence, sixteen fodder species were selected based on their traditional usage to treat diarrhoea and as dietary supplements for cattle. This research aimed to evaluate the nutritional composition and anti-nutritional factors as well as in vitro antibacterial, anti-biofilm, antioxidant, cytotoxicity, and anti-inflammatory activities of plant extracts against E. coli isolates from cattle and reference strains using molecular and phenotypic methods were investigated. The leaves and pods of leguminous (Vachellia, Senegalia, Dichrostachys, Ceratonia, Leucaena) and non-leguminous (Grewia, Morus, Salix, Searsia, Ziziphus) fodder species were collected in the Onderstepoort area, Pretoria North, Gauteng. ICP-MS (Inductively Coupled Plasma Mass Spectrometry), ICP-OES (ICP-Optical Emission Spectrophotometry) and standard methods were utilized to measure trace elements, proximate composition of fodder and tannins. Preliminary screening for antibacterial, antibiofilm (prevention 0 h and eradication 24 h), anti-aggregation and anti-motility activity against three ATCC strains (295225, 35218 and O157:H7) and cytotoxicity against Vero and Caco-2 cells was conducted to select the most active plant extracts for further analysis. Quorum sensing ability was explored using the biosensor strain Chromobacterium violaceum ATCC 12472. Thereafter, characterization of the isolates according to their E. coli UID A housekeeping gene, pathotypes EAEC (pCVD4), EPEC (eae), ETEC (st), EHEC (stx), DAEC (afa B-C), and detection of virulence genes such as agn43/flu, flic, luxS, flix A was undertaken using multiplex PCR. antimicrobial susceptibility testing (AST), anti-biofilm and anti-motility were investigated against ten E. coli strains isolated from healthy cattle faeces against different commercial antibiotics and active plant extracts from the preliminary screening. The seven plants that had good antibiofilm activities were selected for the determination of anti-inflammatory and antioxidant activities using colorimetric tests and qPCR. UPLC/MS/MS analysis was carried out to identify possible active phytochemicals responsible for anti-inflammatory and antibacterial activities of the methanol and acetone extracts of E. rigida. The results highlighted the presence of adequate levels of essential elements such as Zn (21.20-58.00 mg/kg), Co (0.06-3.75 mg/kg), Cr (0.5-5.08 mg/kg), Mn (9.02-197 mg/kg), Mg (0.10-1.08 mg/kg), Fe (42.40-812 mg/kg) and Na (72.00-1721 mg/kg). Notably, toxic elements including were found to be below toxic levels. Proximate analysis revealed the presence of dry matter (> 90%), ash (3.77-26.98%), CP (8.22-22.19%), carbohydrates (54.00-86.79%), fats (0.62- 4.53 mg/mg DM) crude fibre (10.54-40.10%) and neutral detergent fibre (NDF=22.26-59.20%). The gross energy (GE) ranged from 100 to 161.33 MJ/kg DM for most of the selected legume species. Lastly, tannin was present at a safe level (< 50 mg/kg) for ruminant consumption. The antibacterial results revealed that all plants had weak antibacterial activity (MIC > 0.62 mg/ml) with relatively low cytotoxicity (LC50 > 0.02 mg/ml). Approximately 28% of plant extracts eradicated established biofilms (24 h) by more than 50%. Interestingly, both methanol and acetone extracts of E. rigida were the only extracts able to prevent biofilm formation and to eradicate pre-formed biofilm by >50%, hence it was selected for further analysis. The half-maximal concentration of extracts inhibiting violacein production (ranging from 0.01 to 0.002 mg/ml) in all the selected plants with V. erioloba acetone exhibited the best results. E. coli O157:H7 is a hydrophobic and autoaggregating and highly motile organism, but these properties were significantly (p< 0.05) altered after treatment with plant extracts at a sub-MIC concentration of 0.16 mg/ml. Consequently, most plant extracts were inactive in decreasing the EPS production by altering polysaccharides, but the acetone extract of V. nilotica and S. galpinii slowed EPS production by 24% and 28% respectively. Molecular analysis results confirmed that 100% of the bacterial isolates were E. coli strains which were multidrug-resistant against ampicillin (100%), amoxicillin (40%), and tetracycline (30%). The E. rigida methanol extract was non-cytotoxic to Caco2 cells and showed good antibacterial activity against six E. coli isolates with MIC ranging from 0.16 to 0.31 mg/ml compared to acetone (1.25 to 2.5 mg/ml). The E. coli isolates did not harbor any selected genes associated with five diarrhoeal pathotypes including more importantly, the STEC, however, expressed presence of biofilm formation genes (fliA, fliC, (Ag43/flu)) and quorum sensing (lux). All strains were non-aggregative using the SAT test and highly motile with zones of migration < 20 mm after 48 h. E. rigida extracts had motility inhibitory effects in dose dose-dependent manner against E. coli isolates after 48 h incubation. Acetone and methanolic extracts of E. rigida (at 1 mg/ml) inhibited biofilm formation of the E. coli isolates by more than 50%. Seven plants with good antibiofilm formation activity against E. coli ATCC strains were further investigated for their anti-inflammatory and antioxidant activity. The methanol extracts of E. rigida V. tortilis and V. sieberiana inhibited NO release with IC50 values of 90.11, 101.52, and 94.11 μg/ml respectively in comparison to quercetin with IC50 = 30.00 μg/ml. Moreover, these extracts had high LC50 ≥ 70 μg/ml and were therefore relatively non-toxic to LPS-induced RAW 264.7 macrophages. E. rigida, V. sieberiana and V. tortilis extracts decreased the expression of iNOS, COX and IL-6 in LPS-activated macrophages in the same manner as the positive control quercetin. There was a positive correlation between iNOS mRNA expression and NO inhibition by the plant extracts and positive control quercetin. The plant extracts showed very strong antioxidant activity by ABTS and DPPH assays, and strong activity against 15-LOX with low IC50 values of 0.80 and 0.64 mg/ml respectively. UPLC-MS/MS analysis using both negative and positive ionization modes revealed the presence of (1) quercetin-3-O-α-L-rhamnopyranosyl-(1-->6)-β-D-galactopyranoside (609 m/z), (2) eupatin (359 m/z), (3) vicenin (m/z 293) (4) 9-oxo-10,12-octadecadienoic acid (293 m/z) and (5) isorhamnetin-3-O-rutinoside (623 m/z) in acetone and methanol extracts. These phytochemicals are classified as flavonoids and fatty acids which commonly have antimicrobial, antioxidant and inflammatory activities. In conclusion, the present study demonstrated that the methanolic extract of E. rigida, containing flavonoid compounds, essential trace elements (zinc and copper) and crude proteins, may be further investigated as supplementary animal feed or as a phytogenic feed additive because of its useful pharmacological activities including anti-diarrhoeal, antimicrobial, and anti-biofilm activities against multidrug resistant E. coli pathogens. The plant extracts were not toxic to mammalian cells at the highest concentration tested, but animal studies are required to confirm their efficacy because in vitro activity does not translate to in vivo potential. Moreover, the E. rigida extract has the potential to be formulated into antimicrobial surface cleaners and disinfectants to eradicate biofilms on various surfaces such as those in meat processing facilities to avoid contamination of human products.
Clinical signs, lesions and viral antigen distribution in alpacas (Vicugna pacos) naturally infected with Rift Valley fever virus
(University of Pretoria, 2024-06) Odendaal, Lieza; Davis, A. Sally; tasneemanthony@yahoo.com; Anthony, Tasneem
Rift Valley fever (RVF) is a mosquito-borne zoonotic disease affecting both humans and ruminants, causing considerable morbidity and mortality, particularly during the rainy season in Africa. RVF virus (RVFV) infection was first confirmed in camelids in 1962 in Kenya. At the Western Cape Provincial Veterinary Laboratory, several alpaca mortalities were investigated during the 2010-2011 RVF outbreak in South Africa, using histopathology and immunohistochemistry (IHC). Seventeen alpacas were available for this study, with 13 alpacas positive for RVFV-infection by PCR and IHC testing. The 4 PCR- and/or IHC-negative cases were used as controls. In this retrospective study, clinical signs, lesions and viral antigen distribution in alpacas naturally infected by RVFV were documented. Additionally, the tissue and cell tropism in alpacas was studied using immunohistochemistry. Clinically, the majority of the alpacas presented with sudden death with no prior clinical signs. Some of the alpacas had a history of pyrexia, which rapidly progressed to recumbency, depression, respiratory distress, anorexia, and weakness. Unlike lesions typically described for sheep and cattle, macroscopically, the most significant lesions were pulmonary oedema and congestion accompanied by mild to severe ascites. In the majority of the alpacas cases the macroscopic lesions in the liver resembled those of young lambs infected with RVFV, where the necrotic foci were masked by congestion. In RVFV-infected alpacas, microscopic lesions were most consistently present in the liver and lungs and characterized by random, multifocal to extensive and coalescing, hepatic necrosis and pulmonary oedema and congestion. In affected areas in the liver, hepatocytes were swollen or had features typical for apoptosis (Councilman bodies) namely loss of adhesion between cells, condensed hypereosinophilic cytoplasm, chromatin condensation and pyknotic or karyolytic nuclei. Inflammation was minimal with only sparse neutrophils and Kupffer cells in the foci of hepatic necrosis. Comma-or rod-shaped eosinophilic intranuclear inclusion bodies and apoptosis (Councilman bodies) were readily observed in hepatocytes of all RVFV-infected alpacas. In sections of the lungs, there was moderate protein rich alveolar oedema with occasional intravascular macrophages. Only one case had interlobular septal oedema. Immunolabelling for RVFV-positive cases were most consistent throughout the liver samples, followed by the adrenal glands. Labelling was less frequently observed in the spleen, kidney, small intestine, lung, skin and lymph nodes. In these organs, viral antigen was in the cytoplasm of hepatocytes, adrenocortical cells, keratinocytes, large mononuclear cells in the spleen and lymph node most characteristic of macrophages, isolated epithelial cells in the proximal convoluted tubules and thin descending loop of Henle in the kidney, and in endothelial and smooth muscle cells in arterioles and small to medium sized blood vessels of the kidney and lungs. Viral antigen was also in monocytes and intravascular fragments or debris throughout various organs and extracellularly within capillaries in the lamina propria of the small intestines. There was no detectable labelling in the brain and thymus. NovaRed chromogen precipitant was present within the myelin sheaths in the white matter of the cerebrum and cerebellum in 2 of the RVFV-infected cases where brain tissue was available. This was interpreted as non-specific labelling since it was also present in the brain of all the RVFV-negative control cases. In the brain of one of the control cases, there was moderate mononuclear perivascular cuffing, neuronal necrosis, and scattered gliosis of small to medium sized blood vessels. There was also RVFV-positive immunolabelling in the cytoplasm of neurons. This finding was similar to brain lesions and labelling previously reported in alpacas that died following RVF Smithburn vaccination. Therefore, this specific case was likely not a natural infection with wild-type RVFV, but probably due to vaccination with Smithburn vaccine. Clinically alpacas respond like young lambs in that they show minimal clinical signs and die within 12 to 24 hours. Also, like young lambs, the macroscopic lesions in the liver of RVFV-infected alpacas may be obscured by congestion or haemorrhage. Therefore, a diagnosis of RVFV-infection will likely be missed during the initial clinical and post-mortem investigation. However, the microscopic lesions and immunolabelling for RVFV in the liver, adrenal glands, lungs, and spleen are similar to that of sheep. Additionally, apoptotic bodies and eosinophilic intranuclear inclusion typical for RVF were prominent in the liver of all 13 alpacas examined in this study. Therefore, an early diagnosis of RVFV-infection in alpacas is possible based on histology and IHC if tissue samples of the liver are available for examination. If adrenal glands, lungs, and spleen are also available for histological examination the diagnosis is virtually certain and confirmation using another diagnostic test a formality.
A study of the transmission pathways of organisms associated with nosocomial infections at a veterinary academic hospital
(University of Pretoria, 2023) Qekwana, Daniel Nenene; Oguttu, James Wabwire; Kock, Marleen; u26235286@tuks.co.za; Sebola, Dikeledi Carol
Background: Hospital-acquired infections (HAIs) are a major concern in human and veterinary medicine. They are caused by bacterial organisms mainly from the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). Within the veterinary settings, this group of organisms is often zoonotic and tends to acquire resistance determinants. As a result, most of these bacteria are multidrug-resistant, which limits treatment options and patient prognosis. Organisms associated with HAIs are transmitted mainly through the hands of healthcare workers (HCWs), making hand hygiene the most effective measure to prevent and control infections in healthcare facilities. However, low compliance to hand hygiene has been reported in both human and veterinary hospitals, which increases the risk of transmission of HAIs and zoonotic organisms. To reduce the risk of transmission, a multimodal approach has been recommended. As such, this study aims to use a multimodal approach to assess the pre-intervention required to reduce the transmission of organisms associated with HAIs and zoonotic diseases at a veterinary academic hospital. Methods: In order to achieve the objectives of this study, a systematic literature review using the PRISMA method was undertaken to describe the organisms responsible for HAIs and zoonotic infections. In addition, antimicrobial resistance genes associated with these organisms were also described. Since the results of the systematic literature review showed there was limited information on the burden of ESKAPE pathogens in South Africa, data on dog clinical cases presented at the veterinary academic hospital between 2007 and 2013 were reviewed. Klebsiella pneumoniae and A. baumannii isolates were assessed in terms of their burden and antimicrobial resistance patterns. Hands of healthcare workers were also assessed for the presence of organisms associated with HAIs and zoonotic diseases using the polymerase chain reaction (PCR). In addition, each isolate was subjected to antimicrobial sensitivity testing following the Kirby-Bauer disk diffusion method. In order to assess the level of knowledge of veterinary students regarding the transmission of HAIs, a questionnaire survey was performed assessing the knowledge of students on infection prevention and control (IPC) and the transmission of organisms associated with HAIs. Results: Bacterial organisms associated with HAIs and zoonosis were reported from clinical cases, environmental surfaces, and items used during patient treatment and care. Staphylococcus species was the most reported organism, and some isolates seem to share similar clonal lineage to those reported in humans. In terms of resistant genes, the mecA gene was identified in both Methicillin- resistant Staphylococcus aureus (MRSA) and Methicillin- resistant Staphylococcus pseudintermedius (MRSP), the blaCMY-2 gene in E. coli and Salmonella spp., flo genes in E. coli, and the vanA gene in E. faecium isolates. Acinetobacter baumannii (n=20) and K. pneumoniae (n=56) isolates were isolated from bronchoalveolar lavage, foreign objects, bone, urine, skin, blood, ear, nasal, and oral cavity. Sixty percent (60%) of A. baumannii were multidrug-resistant (MDR) while 98% were MDR K. pneumoniae. Of the students tested (62), at least one of the ESKAPE pathogens were isolated from their hands. Escherichia coli was the most isolated (76%, 47/62), followed by E. faecium (52%, 22/62), P. aeruginosa (48%, 30/62). A. baumannii (47%, 29/62), K. pneumoniae (27%, 17/62), and S. aureus (24%, 15/62). Resistance to at least one antibiotic was high among E. coli isolates (100%, 9/9), followed by E. faecium (67%, 4/6), P. aeruginosa (100%, 13/13), A. baumannii (57%, 4/7), K. pneumoniae (100%, 7/7), and S. aureus (67%, 2/3). Only E. coli (42%, 5/12), E. faecium (40%, 2/5), P. aeruginosa (100%, 13/13), and S. aureus (33%, 1/3) were multidrug resistant. Of the 147 students interviewed most were female (69%, 102/147) followed by male (29%, 43/147). Two (1%, 2/147) students did not indicate their sex. Less than half (41%, 60/147) of the respondents indicated they heard about IPC practices. However, they were aware that jewellery, stethoscopes, ward telephones, and leashes are possible sources of pathogens associated with HAIs. Conclusion: Bacterial organisms associated with hospital-acquired and zoonotic diseases were reported from clinical cases, environmental surfaces, and items used in veterinary service. The hospital environment where there is human contact had the highest burden of organisms associated with HAIs. Moreover, the ESKAPE organisms were identified in the hands of the students working in the ICU. Organisms associated with HAIs in this study were often MDR which is likely to impact patient care and prognosis. In addition, if contaminated, students would likely pass on these pathogens to other persons and animals. The results of this study further support suggestions that human behaviour plays a crucial role in the transmission of HAIs in veterinary hospitals. The study also shows from the survey that students do not have a good understanding of IPC measures and their role in the prevention of HAIs and zoonotic diseases although taught during lectures.
Respiratory, cardiovascular and metabolic effects of etorphine, with and without butorphanol, in white Rhinoceros (Ceratotherium Simum)
(University of Pretoria, 2019-11) Meyer, L.C.R. (Leith Carl Rodney); Buss, Peter Eric; Gleed, Robin D.; jmb264@cornell.edu; Boesch, Jordyn Marie
This thesis presents data that increases our understanding of the pathophysiological effects of etorphine in the white rhinoceros (Ceratotherium simum). This iconic species is poached for its horn throughout southern Africa and must be managed strategically for its protection. Management procedures require chemical immobilisation, and the ultrapotent µ (mu) opioid receptor agonist, etorphine, is one of the few drugs available for rhinoceros immobilisation. However, since etorphine was first introduced in the mid-20th century, veterinarians have observed severe side effects in white rhinoceros during immobilisation, the most serious of which is, arguably, hypoxaemia. Although uncommon, mortalities have occurred.1 An understanding of the mechanisms by which etorphine causes these derangements will inform efforts to develop preventative or therapeutic strategies that will increase the safety of chemical immobilisation for the white rhinoceros, thus contributing to its conservation. Previous studies have evaluated heart rate (fH), systemic arterial pressure (SAP), arterial blood gases and acid-base status, minute ventilation (VE), plasma catecholamine concentrations, metabolic rate, and tremors, among other variables.2-7 However, a comprehensive understanding of the effects of etorphine requires measurement of pulmonary pressures, cardiac output (Qt), and mixed venous blood gases and acid-base status as well. To this end, my colleagues and I developed and refined an ultrasound-guided technique for pulmonary arterial catheterisation in the white rhinoceros. In other species, pulmonary arterial catheters (PAC) are inserted through a percutaneous introducer into the jugular vein; the tip is passed through the right heart and into the pulmonary artery. Ultrasound examination suggested that percutaneous access to the jugular vein is impractical in white rhinoceros. However, my colleagues and I found that in this species the introducer can be inserted instead into the linguofacial vein, a branch of the jugular vein, and designed a PAC long enough to reach the pulmonary artery. Pilot testing of a custom-built PAC in boma-habituated white rhinoceros immobilised with etorphine and azaperone, followed by butorphanol intravenously (IV), afforded me the opportunity to assess some of the cardiopulmonary effects of a supplemental, IV bolus of etorphine. I observed an increase in mean pulmonary arterial pressure (mPAP), Qt, and fH and a decrease in arterial oxygen partial pressure (PaO2) following an etorphine bolus. Based on normal values in the white rhinoceros and the horse, or values calculated allometrically for the white rhinoceros, these variables were already pathologically increased (mPAP, Qt, fH) or decreased (PaO2), and the bolus worsened these physiological derangements. The development and refinement of the pulmonary arterial catheterisation technique, and the data on the effects of an etorphine bolus are detailed in Chapter 2. After increasing the PAC length to enable measurement of pulmonary arterial occlusion pressure (PAOP), my colleagues and I conducted further research in six boma-habituated, sub-adult, male white rhinoceros. Using a crossover design, each rhinoceros was administered each of two treatments (i.e., two study phases) in random order: etorphine intramuscularly (IM) followed by saline IV (treatment ES), or etorphine IM followed by butorphanol IV (treatment EB). Once a rhinoceros was positioned in lateral recumbency (time = 0 minutes [t = 0]), 30 minutes was allotted for instrumentation, which included pulmonary and systemic arterial catheterisation and connection to a custom breathing system. Baseline data were collected at t = 30, saline or butorphanol was administered at t = 37, and data were collected again at t = 40 and 50. Chapter 3 presents data from ES and examines changes over time. As reported in previous studies, etorphine produced severe hypoxaemia and hypercapnia, and VE was lower than normal, expected VE calculated allometrically.2 I found that over time, physiological variables remained relatively constant after etorphine administration, with a few exceptions. Mixed venous oxygen partial pressure (PῡO2), arterial oxygen content (CaO2), and mixed venous oxygen content (CῡO2) increased (i.e., improved); tidal volume (VT) decreased (i.e., worsened), and tremor score and mixed venous lactate concentration decreased (i.e., improved). Oxygen consumption (VO2) initially decreased (i.e., improved) then increased somewhat. (However, some of these differences lost significance after correction for multiple comparisons; refer to Chapters 3 and 4.) Values for mPAP, PAOP, mean SAP (mSAP), Qt, VO2, and plasma noradrenaline concentrations were higher than ‘normal’, assessed by comparison to values measured in other species (or those calculated allometrically), consistent with sympathetic outflow. Chapter 4 examines changes over time in EB and compares ES and EB. I found that butorphanol decreased (i.e., improved) pulmonary pressures, mSAP, Qt, fH, pulmonary artery and rectal temperatures (T), arterial carbon dioxide partial pressure (PaCO2), VO2, carbon dioxide production (VCO2), oxygen extraction ratio (OER), shunt fraction (Qs/Qt), noradrenaline concentration, tremor score, and haemoglobin (Hb) concentration ([Hb]); it also decreased (i.e., worsened) VT. Butorphanol increased (i.e., improved) pulmonary vascular resistance (PVR), PaO2, PῡO2, CaO2, CῡO2, minute ventilation body temperature and pressure, saturated with water vapour (VEBTPS), and respiratory rate (fR). These differences were either changes from baseline in EB or differences at one or more sampling points between ES and EB.
Inhibition of biofilm formation of foodborne pathogens by selected South African medicinal plants
(University of Pretoria, 2019-08) McGaw, Lyndy Joy; chineloerhabor@gmail.com; Erhabor, Chinelo Rosemary
Microbial biofilm and quorum sensing are related traits employed by microorganisms to improve their survival and virulence. They have been increasingly implicated in the food processing and the medical industries where they cause surface and food surface contamination. In this thesis, the available literature regarding the value of South African plants as potential sources of anti-biofilm and quorum quenching bioactive secondary metabolites was surveyed. The literature survey also covered antimicrobial activity investigations of medicinal plants against foodborne pathogens. The survey revealed that a total of thirty plant species belonging to nineteen families have anti-biofilm and quorum quenching capacity against foodborne pathogens. The survey served to summarize present knowledge and to provide a basis for further investigation of South African medicinal plants with known anti-biofilm and quorum quenching potential. In subsequent research, the antimicrobial, anti-biofilm, antioxidant and cytotoxicity activities of nine South African medicinal plants was evaluated. The plants (Combretum elaeagnoides, Combretum molle, Combretum oxystachyum, Carpobrotus edulis, Vachellia rehmanniana, Vachellia xanthophloea, Kigelia africana, Elephantorrhiza elephantina and Ochna pretoriensis) were investigated. The selection of plant species was based on their known antimicrobial activity, chemotaxonomic relationships to plant species with antibacterial activity, availability and/or the existence of traditional uses against foodborne diseases. The serial microdilution technique and the crystal violet assay were used to assess the antimicrobial and anti-biofilm potential of the acetone and methanol extracts, fractions and isolated compound. The antioxidant activity of the extracts, fractions and isolated compound was determined using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assays. The tetrazolium dye cell viability assay was used to ascertain the cytotoxicity of the samples. The methanol extract of C. elaeagnoides was fractionated into five fractions (hexane, dichloromethane, ethyl acetate, butanol and water) via solvent-solvent fractionation. Column chromatography making use of silica gel as stationary phase combined with gradient elution of increasing polarity using hexane and ethyl acetate was used to isolate a bioactive compound via bioassay-guided fractionation. The isolated compound was identified as quercetin 3-O-rhamnoside using mass spectrometry (MS) and nuclear magnetic resonance (NMR). The extracts of the nine plants were active against all selected test organisms with minimum inhibitory concentration (MIC) values ranging from <0.02 to 2.5 mg/ml. The acetone extracts of Vachellia xanthophloea, Combretum molle, and Carpobrotus edulis had excellent MIC activity of <0.02 mg/mL against Enterobacter cloacae. The dichloromethane fraction of Combretum elaeagnoides had the best MIC value of 0.03 mg/mL against Escherichia coli (ATCC 25922). The result of the minimum bactericidal concentration (MBC) investigation showed that the acetone extracts of C. elaeagnoides and C. molle had the best activity against E. coli (ATCC 25922) with no growth at 0.31 mg/mL, and acetone extracts of C. elaeagnoides and V. xanthophloea against Staphylococcus aureus at 0.63 mg/mL respectively. The acetone extracts of C. molle and V. xanthophloea had the best MBC activity against the clinical isolate of E. coli (clinical isolate), Campylobacter jejuni, and E. cloacae with 100% inhibition at 0.08, 0.31 and 0.02 mg/mL respectively. The acetone extract of C. edulis also inhibited (100%) E. cloacae at 0.02 mg/mL. The acetone extract of C. molle exhibited 100% inhibition against Salmonella Typhimurium, Stenotrophomonas maltophilia and Klebsiella pneumoniae at 0.04, 0.16 and 0.08 mg/mL respectively. The acetone extract of V. xanthophloea also exhibited 100% inhibition against Salmonella Typhimurium and S. Enteriditis at 0.08 mg/mL. Of all the extracts, fractions and the isolated compound, the acetone extract of C. molle had the best MBC against the tested pathogens. Most of the extracts, fractions and the isolated compound, quercetin 3-O-rhamnoside, selectively reduced biofilm growth by at least 50% against the foodborne pathogens. The acetone extract of C. molle had the greatest anti-biofilm activity against S. Typhimurium. The antioxidant assay revealed that the methanol extract of V. xanthophloea had very good activity against the radical scavenging DPPH while the acetone extract of C. edulis had excellent activity against the electron reducing ABTS with IC50 values of 0.14±0.11μg/mL and 0.01±0.02 μg/mL respectively. Most of the fractions had good antioxidant activity against DPPH (IC50= 0.01 to >100 μg/mL) and ABTS (IC50= 0.01±0.007 to 20.00±1.79 μg/mL) radicals. The butanol fraction of C. elaeagnoides and quercetin 3-O-rhamnoside had excellent antioxidant activity with the same IC50 value of 0.01 μg/mL. The cytotoxicity assay revealed that most of the extracts were relatively safe to cells except for the acetone extract of C. molle (0.01 mg/mL) and the methanol extract of O. pretoriensis (0.02 mg/ml). Therefore the good antimicrobial activity of C. molle may be largely owing to non-specific toxicity. In summary, the study has established that the leaf extracts of the nine South African medicinal plants as well as the fractions and isolated compound from C. elaeagnoides have antimicrobial, antibiofilm and antioxidant potential and are generally relatively non-cytotoxic. Further work is needed to explore the quorum quenching, synergistic effects and possible mechanisms of action of the plants.
Hand hygiene compliance in the intensive care unit of the Onderstepoort Veterinary Hospital, South Africa
(University of Pretoria, 2019-10) Qekwana, Daniel Nenene; Boucher, Charles; dc.sebola@gmail.com; Sebola, Dikeledi Carol
Infection prevention and control (IPC) practices play an important role in the prevention and management of hospital-acquired infections (HAIs) in healthcare facilities and hand hygiene is the cornerstone of all IPC practices. Despite the effectiveness of IPC in the management of HAIs, its adoption in veterinary medicine has been limited. Additionally, there is paucity of data on IPC practices in veterinary medicine. Therefore, this study evaluated hand hygiene compliance among healthcare workers in the intensive care unit (ICU) at the Onderstepoort Veterinary Academic Hospital. A cross-sectional study was conducted among healthcare workers (HCWs) and visitors in the ICU. The infection control assessment tool (ICAT), focusing on the five hand hygiene moments criteria was used to evaluate compliance. The level of compliance and a 95% confidence interval was calculated for all variables. Individual bottles of alcohol-based hand rub solutions and hand-wash basins with running water, soap dispensers, and paper towels were available in the ICU. In total, 296 observations consisting of 734 hand hygiene opportunities were recorded. In addition, hand hygiene compliance was also evaluated during invasive (51.4%) and non-invasive (48.6%) procedures. Most HCWs did not sanitize stethoscopes, leashes, and cellular phones used in between patients. Additionally, the majority of them were not bare “below the elbows” because they wore jewellery. The overall hand hygiene compliance was 24.3% (178/734). The most common method of hand hygiene was hand rub (58.4%) followed by hand-wash (41.6%). Nurses had a higher (44%) level of compliance compared to students (22%) and doctors (15%). Compliance was also higher after body fluid exposure (42%) compared to after patient contact (32%), before patient contact (19%), after contact with patient surroundings (16%), and before an aseptic procedure (15%). Furthermore, nurses had the lowest compliance after body fluid exposure (14%), students had the lowest compliance before patient contact (16%) and doctors had the lowest compliance after contact with patient surroundings (0%). The low levels of hand hygiene compliance in this study raises concerns of potential transmission of HAIs and zoonosis in the ICU. Therefore, intervention strategies are recommended to improve the compliance level in the hospital. These may include an educational campaign on the importance of adhering to hand hygiene, development and promotion of written hand hygiene protocols, and programs promoting regular hand hygiene auditing could be developed.
Controlling Haemonchus contortus using bioactive compounds from plants with antifungal activity
(University of Pretoria, 2020-02) McGaw, Lyndy Joy; Eloff, Jacobus Nicolaas; Naidoo, Vinny; belosakong@gmail.com; Sakong, Bellonah Motshene
Infection of the gastrointestinal tract of livestock by the nematode parasite Haemonchus contortus is a serious challenge to livestock production. Infected animals, especially sheep and goats, frequently suffer from diarrhoea, anaemia, loss of appetite, reduced body weight and ultimately death if untreated. Anthelmintic drugs are widely used to control nematode infection, but problems of the development of resistance by parasites and unavailability or cost to rural smallholder farmers are serious problems. Their use has also resulted in the presence of residues in meat and milk, which affects food safety. Therefore, alternative control measures such as using plant extracts with putative anthelmintic properties can enhance parasite management. The aim of this study was initially to investigate plant species used in ethnoveterinary medicine to control intestinal parasites for in vitro anthelmintic activity against the sheep nematode Haemonchus contortus. These extracts did not give promising results as the anthelmintic activities were generally low so further work on these plant species was discontinued. The second part of the project was to test plant species with known antifungal activity for activity against H. contortus and a panel of fungal species, as some anthelmintic chemicals also have antifungal activity. For example the benzimidazole group of drugs has demonstrated good correlation between antifungal and anthelmintic activity. Antiparasitic activities of the acetone and water extracts of six plant species with known antifungal activity were determined using the egg hatch assay recommended by the World Association for the Advancement of Veterinary Parasitology (WAAVP). Cytotoxicity was determined by evaluating the viability of cells in the presence of the plant extracts using the tetrazolium-based colorimetric assay against Vero African Green monkey kidney cells. Efficacy of the extracts against various fungi was also tested using a serial microdilution method against three fungal pathogens (Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans), as well as plant fungal pathogens. As parasitic anthelmintic assays are laborious and difficult, a major objective of this study was to investigate if a simple and rapid antifungal assay can be used as a model to isolate anthelmintic compounds from plant extracts. The extracts of Diospyros whyteana and Peddiea africana had the strongest egg hatch inhibitory potential. The extracts had relatively low toxicity to normal mammalian Vero cells. Water extracts were slightly more toxic than acetone crude extracts. All the extracts had good antifungal activities with minimum inhibitory concentration (MIC) values as low as 0.04 mg/ml. Peddiea africana, followed by Schotia brachypetala extracts had the best antifungal activity. Schotia brachypetala inhibited the fungi at 0.04-0.08 mg/ml and the cytotoxicity was low, leading to selectivity indices ranging from 0.59-13.43. The water extract of Cassipourea gummiflua was the most active of all the water extracts. The acetone extracts had a higher number of bioactive compounds than water extracts based on the number of fungal inhibition areas in bioautography. In the anthelmintic egg hatch assay (EHA), the Diospyros whyteana acetone leaf extract had good activity. The extract was also active against Candida albicans with an MIC of 0.04 mg/ml. Candida albicans was therefore used as a model for bioassay-guided fractionation to isolate antifungal compounds from D. whyteana. Successive column chromatography resulted in isolation of three active compounds. Two compounds were not isolated in sufficient quantity to allow structural elucidation and identification but the third compound was identified as a mixture of α-amyrin and β-amyrin. This mixed compound had good antifungal (MIC values as low as 40 μg/ml) as well as anthelmintic activity (30-50% inhibition of egg hatch inhibition of H. contortus). This validates the use of the antifungal assay in bioassay-guided fractionation to isolate anthelmintic compounds in this case. Additional useful activities for anthelmintic or antifungal plant-based remedies include antioxidant activity which may assist the patient in combating the disease. The total phenolic and flavonoid contents of the plant species were investigated, together with antioxidant activity using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. The total phenolic content of the extracts of Diospyros whyteana (water extract) was the highest followed by Schotia brachypetala (water extract). Generally, water extracts exhibited higher total phenolic contents than the acetone extracts. The plant extracts also generally had relatively high flavonoid contents. Phenolic and flavonoid compounds are often associated with antioxidant activity. The DPPH radical scavenging antioxidant activity of the extracts was less potent than those of the positive controls ascorbic acid and trolox. The acetone extracts of Peddiea africana and Schotia brachypetala had good DPPH radical scavenging ability. This study showed that extracts of some plant species with good antifungal activities also have anthelmintic activity. This may indicate common biological pathways of inhibition or mechanisms of action worthy of further investigation. The use of the antifungal assay in this study assisted with the isolation of anthelmintic compounds. Diospyros whyteana had good anthelmintic and antifungal activity, and compounds active in both assays were isolated and identified from the acetone extract of this species. Further work on other plant species is necessary to confirm the correlation of antifungal and anthelmintic activity in plant extracts.
Physiological responses to capture and transport in southern white rhinoceros (Ceratotherium simum simum) and southern-central black rhinoceros (Diceros bicornis minor)
(University of Pretoria, 2019) Meyer, L.C.R. (Leith Carl Rodney); Hooijberg, Emma H.; Buss, Peter Eric; friederike.pohlin@gmail.com; Pohlin, Friederike
The southern-central black rhinoceros is currently listed as “critically endangered” and the southern white rhinoceros as “near threatened” by the International Union for Conservation of Nature Red List of Threatened Species. Translocation is an important tool for rhinoceros conservation and is widely used to reinforce declining populations or to restore extirpated populations. Capture and transport are part of translocation and expose the rhinoceros to a variety of stressors that ultimately can lead to morbidity and mortality. The broad objectives of this thesis were therefore to: (1) establish a better understanding of the physiological responses to capture and road-transport in African rhinoceros, (2) to identify challenges associated with transport that should be addressed in order to improve animal welfare, and (3) to investigate whether the use of midazolam, instead of azaperone, would be able to mitigate some of these challenges. The third chapter of this thesis investigated physiological responses to capture and long road transport in black and white rhinoceros in a “real-world” setting. Paired venous blood samples were collected from an auricular vein at capture and after transport in 14 boma-adapted black, and 32 semi-captive white rhinoceros and were evaluated for changes in: (1) clinical chemistry analytes; (2) acute phase reactants and (3) oxidative stress biomarkers. The Wilcoxon rank sum test was used to compare changes in measured analytes from capture and after transport. The fourth and fifth chapter explored some of these changes in more detail and investigated if there were differences between rhinoceros sedated with midazolam compared to azaperone. Twenty-three wild white rhinoceros bulls were captured with a combination of etorphine plus either azaperone or midazolam. Azaperone or midazolam, respectively, were re-administered intramuscularly at capture (TC), start of transport (T0), and at two (T2) four (T4) and six (T6) hours of transport. Acid-base status was evaluated at these time points. Adrenaline and cortisol concentrations, as well as haematological (erythron, thrombon, leukon) and immunological (leukocyte coping capacity, acute phase reactants and oxidative stress biomarkers) changes, were measured at TC, T0 and T6. Changes in measured variables over time and between groups were compared using general mixed effects models. Black and white rhinoceros transported over a long time experienced total body water loss, mobilisation of energy reserves, muscular damage, and stress-induced immunomodulation. White rhinoceros bulls experienced respiratory acidosis combined with a lactic- and non-volatile weak acid acidosis during capture, followed by a mild metabolic- and strong ion alkalosis during transport. Increases in plasma adrenaline and serum cortisol concentrations indicated that rhinoceros mounted a stress response to capture and transport. The stress response was associated with characteristic haematological and immunological changes including stress-haemoconcentration, a progressive increase in neutrophil to lymphocyte ratio, the mounting of an acute phase reaction and oxidative stress. The acidaemia and associated alterations in acid-base indices were significantly less pronounced in midazolam-sedated compared to azaperone-sedated rhinoceros. Plasma adrenaline and serum cortisol concentrations did not differ between the groups. Midazolam, however, appeared to greater influence immunological responses to stress than azaperone. Based on these results, we identified the following challenges to animal welfare during rhinoceros capture and transport: (1) life-threatening acid-base abnormalities associated with the unique challenges of rhinoceros capture; (2) fear and the mounting of a stress response to capture and the novelty of transport; (3) stress-induced immunomodulation; and (4) skeletal muscle fatigue, energy imbalance and dehydration that likely become more relevant with transport time. Midazolam proved to be able to partly mitigate the first challenge and may therefore be a safer alternative to azaperone when combined with etorphine for the capture of wild white rhinoceros. Further research looking at behavioural benefits of using midazolam over azaperone, and possible consequences of its immunological effects, are required to demonstrate the value of midazolam administrations during transport. To optimise rhinoceros translocation, additional measures that aim to mitigate challenges to animal welfare during capture and transport, such as administering fluids during long journeys, need to be included in the planning of future translocations. The effectiveness of these measures in mitigating these challenges need to be systematically investigated.
Reference intervals for selected haematological and clinical biochemistry measurands in Temminck’s ground pangolin (Smutsia temminckii)
(University of Pretoria, 2019) Meyer, L.C.R. (Leith Carl Rodney); Hooijberg, Emma H.; karinlourens@gmail.com; Lourens, Karin
An alarming number of pangolins are currently illegally traded for their scales and meat. Many pangolins confiscated from the trade are severely clinically compromised. Unfortunately, little is known about the physiology and normal health of pangolin, making it difficult to identify disease processes and treat them. The purpose of this study was to establish reference intervals (RIs) for haematology and plasma clinical chemistry in the Temminck’s ground pangolin. Blood samples were collected from 27 healthy free-living or rehabilitated pangolins and reference intervals were generated according to international guidelines. Clinical chemistry analysis was performed using the Abaxis VetScan VS2 and Cobas Integra 400 Plus analyser and haematology was performed using the Abaxis VetScan HM5 analyser. Vetscan VS2 plasma clinical chemistry RIs were: albumin 26-41 g/L, amylase 316-1014 U/L, ALP 29-153 U/L, ALT 25-307 U/L, bilirubin 1.5-10.8 mol/L, calcium 1.8-2.5 mmol/L, creatinine 9.7-46.3 mol/L, glucose 3.8-10.0 mmol/L, phosphate 1.3- 2.6 mmol/L, total protein 53-84 g/L, and urea 5.6-19.9 mmol/L. Cobas plasma clinical chemistry RIs were: albumin 19-33 g/L, amylase 396-1669 U/L, ALP 25-301 U/L, ALT 17-291 U/L, bilirubin 1.5-18.3 mol/L, calcium 1.8-2.4 mmol/L, creatinine <58 mol/L, glucose 3.6-10.1 mmol/L, phosphate 0.9-2.3 mmol/L, total protein 48-74 g/L, and urea 6.2-20.4 mmol/L. Haematology RIs were: WBC 1.8-10.71 x109/L, RBC 3.88-8.31 x1012/L, HGB 73-150 g/L, HCT 26-51%, MCV 55-72 fL, MCH 15.6-21.4 pg, MCHC 242-332 g/L, and RDW 14.3-19.1%. The Wilcoxon test revealed significant differences between results for the following measurands for the Cobas versus the Abaxis Vetscan VS2: albumin (p = <0.0001); ALT (p = <0.0001); amylase (p= <0.0001); bilirubin (p= 0.038); calcium (p= <0.0001); phosphate (p= <0.0001); total protein (p= <0.0001); urea (p= <0.0001). RIs for some measurands were wide, probably due to the small sample size. Nevertheless, these are the first RIs generated for the Temminck’s ground pangolin and the results presented here will allow veterinarians to better determine the health status of pangolin patients, thus enabling them to formulate optimal treatment plans in the hope of increasing patient survival rates of this endangered species.
Determining whether blood colour can be used to assess arterial blood oxygenation in immobilised impala (Aepyceros melampus)
(University of Pretoria, 2019) Meyer, L.C.R. (Leith Carl Rodney); Zeiler, Gareth Edward; drpebasson@gmail.com; Basson, Pierre Etienne
Hypoxaemia (oxyhaemoglobin saturation < 90%) often occurs during wildlife immobilisation and poses a risk of morbidity and mortality. Several methods have been used to assess blood oxygenation in immobilised impala (Aepyceros melampus). Pulse oximetry has been shown to be unreliable, co-oximetry and blood gas analysis are the gold standard but are limited by practicality and cost. With the advent of digital cameras and spectrocolourimeters the assessment of blood colour could be of value for determining blood oxygenation. This study set out to determine whether there is good association between arterial blood colour, as assessed by CIE L*a*b* (Commission on international illumination; L*: luminosity; a*: green to red; b*: blue to yellow) colour components, and blood oxygenation, as determined by functional oxyhaemoglobin saturation (SaO2) and fractional oxyhaemoglobin saturation (FO2Hb). To obtain arterial blood samples with different blood oxygen levels 11 impala were immobilised with either etorphine or thiafentanil. Arterial blood samples were collected from the auricular artery at five-minute intervals and immediately analysed by means of co-oximetry to measure blood oxygenation, and spectrocolourimetry to measure the CIE L*a*b* colour components. The colour components associated better with blood oxygenation (SaO2 and FO2Hb) using a quadratic rather than a linear model (p < 0.001). The association was strong for each of the colour components (CIE L*a*b*). Therefore both SaO₂ and FO2Hb are reliable predictors of all three CIE L*a*b* components of arterial blood colour, and hence blood colour can be used to reliably estimate arterial blood oxygenation of impala. These findings could pave the way for developing colour charts and devices that can be used in the field to inexpensively determine blood oxygenation, and detect hypoxaemia, in immobilised or anaesthetised animals.
Efficacy of selected plant species against Meloidogyne incognita and phytopathogenic microbes infecting tomatoes
(University of Pretoria, 2021-07) McGaw, Lyndy Joy; fnmakhubu@gmail.com; Makhubu, Fikile Nelly
Soilborne pathogens are economically important, causing great losses in agricultural production globally. Nematodes, fungi, bacteria and viruses are microscopic, destructive pathogens which are extremely difficult to control. Farmers depend on synthetic pesticides for quick and effective control of pests and diseases. However, the majority of nematicides have been banned due to environmental problems resulting from their use and this has awakened interest in finding alternative methods of nematode and other plant pathogen control. Several indigenous plants have been identified as having potential anthelmintic efficacy in managing gastrointestinal infections in small ruminants and some of these have also been found to be effective in killing nematodes infecting crops. In the present study, extracts of plants previously reported to have anthelmintic activity against free-living Caenorhabditis elegans and animal parasitic Haemonchus contortus nematodes were screened for their efficacy against the root-knot nematode species Meloidogyne incognita. Activity of the selected plants was confirmed against C. elegans and H. contortus. For in vitro safety determination, cytotoxicity tests on Leonotis leonurus, Clausena anisata and Lantana rugosa was conducted against Vero African green monkey kidney cells. Chemical profiling was performed using gas chromatography-mass spectrometry (GC-MS). The plants were further investigated for their ability to inhibit the growth of interrelated phytopathogens infecting tomatoes, including five pathogenic bacterial species: Clavibacter michiganensis subsp. michiganensis, Xanthomonas vesicatoria, X. perforans, Ralstonia solanacearum and R. pseudosolanacearum and one fungal strain (Fusarium oxysporum f.sp. lycopersici). Bioactive compounds were isolated from the acetone leaf extract of L. leonurus using bioassay-guided fractionation. Clausena anisata, L. rugosa and L. leonurus water extracts were subjected to in vitro phytotoxicity tests on tomato seedlings, and an in vivo glasshouse trial against M. incognita was also conducted with the powdered material (dried leaves) of L. leonurus and C. anisata. All plant extracts and fractions investigated had good anthelmintic activity against free-living and animal-parasitic nematodes. Activity of the plant species Acokanthera oppositifolia, Searsia lancea, Cotyledon orbiculata, Hippobromus pauciflorus and Lantana rugosa was reported for the first time against Haemonchus contortus. The phytochemicals detected in the extracts contribute to the activity of the plants reported, as some of these compounds were previously reported to have antibacterial, insecticidal and nematicidal properties. Cytotoxicity results indicated that C. anisata and L. leonurus extracts were relatively non-toxic to Vero cells compared to the positive control doxorubicin (LC50 = 0.0133 mg/mL). Lantana rugosa extracts were highly toxic with LC50 values less than 0.0075 mg/mL, the lowest concentration tested. When screening the selected plants for activity against M. incognita second-stage juveniles (J2) at the highest concentration of 1 mg/ml, results indicated that all selected non-crop plant species had potential in managing root-knot nematodes, but the most promising activity was observed with C. anisata, L. rugosa and L. leonurus extracts. The three plants were further evaluated in terms of motility against J2s as well as J2 egg hatch inhibition. Only the L. leonurus water extract showed good dose-related activity in inhibiting motility of M. incognita J2s. Clausena anisata extracts had weak activity in the motility assay but both plants had good inhibitory activities against J2 hatching. In the antimicrobial assay, acetone extracts of Leucosidea sericea and Searsia lancea demonstrated interesting antibacterial activity against a broad range of tested bacteria with MIC values ranging from 19.5 to 97.5 µg/mL. None of the extracts was active against Fusarium oxysporum f. sp. lycopersici except for the acetone extract of Cotyledon orbiculata and L. leonurus water extract, which inhibited the growth of F. oxysporum f.sp. lycopersici at 39 µg/mL and 97.5 µg/mL after 24 h respectively. Clausena anisata and L. leonurus fractions obtained from liquid/liquid partitioning indicated poor activity against all tested phytopathogens, except for the L. rugosa fractions which had good to moderate activity against most bacterial pathogens with MIC values ranging from 78 to 156 µg/mL. Bioassay-guided fractionation was used to isolate nematicidal compounds from the dichloromethane fraction of Leonotis leonurus using Caenorhabditis elegans as the test organism. This led to isolation of leoleorin C, and other compounds which could not be identified due to insufficient time and purity. Leoleorin C was previously isolated from the plant, but activity against selected organisms and the in vitro lack of cytotoxicity is reported for the first time in this study. Leoleorin C had moderate activity against C. elegans but was not active against M. incognita. The compound was not active against the tested bacterial phytopathogens, but promising activity was observed in the bioautography assay against Clavibacter michiganensis subsp. michiganensis, causal organism of bacterial canker in tomatoes. The plant was not toxic to the Vero cells at the highest concentration of 1 mg/mL. Leonotis leonurus, C. anisata and L. rugosa water extracts were not phytotoxic to the growth of tomato seedlings at all tested concentrations (highest concentration of 10 mg/mL) under in vitro conditions. Lower extract concentrations of all the selected non-crop plant species stimulated germination, whereas high concentrations had inhibitory effects, except for the C. anisata extract. In the treatment of tomato plants under in vivo conditions, M. incognita infection did not negatively affect the growth parameters assessed, namely stem height, wet shoot mass, dry shoot mass, number of flowers, stem diameter, fruit number and fruit mass. The plants had the ability to reduce gall index, number of eggs and J2 when compared to the untreated control of tomato plants. There was a lack of phytotoxic and fertilizer effect on tomato plants at the applied rates on growth parameters since activity was comparable to the untreated control. The low effect demonstrated by the plants might be due to low quantities of active phytochemicals present. Leonotis leonurus and C. anisata were not effective in the form of powder as nematicidal preparations in the glasshouse trials.
Efficacy of selected South African plants against multidrugresistant staphylococci isolated from clinical cases of bovine mastitis
(University of Pretoria, 2021-05) McGaw, Lyndy Joy; Petzer, Inge-Marie; ladeboye@yahoo.com; Ayodele, Omolade Akinboye
Among cattle diseases, bovine mastitis remains a serious problem with financial implications to the farmer as it adversely affects milk production. Staphylococcus aureus and non-aureus staphylococcus (NAS) species are often isolated from milk samples in cases of bovine mastitis. Various antimicrobial agents have been used for treatment of mastitis pathogens in veterinary medicine with limited success because of increasing prevalence of resistance to commonly used antibiotics. Some bacteria causing bovine mastitis are known for their ability to form biofilms, which helps them resist antibiotic effects. The use of natural plant products is being explored as an alternative antimicrobial treatment for mastitis and a template for new mastitis drug development. The primary aim of this study was to investigate antibacterial and antibiofilm activities of selected South African plants against drug-resistant staphylococci organism isolated from clinical cases of bovine mastitis. The study also examined the cytotoxic, anti-inflammatory and antioxidant activities of some of the plants. Plants for this study were selected based on known antimicrobial activity, chemotaxonomic relationships to plant species with antibacterial activity, availability, and/or the existence of traditional uses against infectious diseases. The nine selected South African plants include Antidesma venosum, Elaeodendron croceum, Erythrina caffra, Indigofera frutescens, Pleurostylia capensis, Searsia lancea, Searsia leptodictya, Trichilia emetica and Ziziphus mucronata. Eight S. aureus isolates, three Staphylococcus chromogenes isolates, one Staphylococcus haemolyticus isolate and one ATCC strain of S. aureus were used to determine the antibacterial activity of extracts of the plants prepared with acetone and ethanol using standard methods, while one S. aureus isolate and one ATCC strain of Staphylococcus epidermidis were used in the antibiofilm assay. The leaf extracts of the plants with good antimicrobial activity were also assessed for their antibiofilm, antioxidant, anti-inflammatory and cytotoxic activities using standard methods. The range of antibacterial minimum inhibitory concentration (MIC) values obtained in this study was relatively low and promising. All the plant extracts had good to weak antibacterial activity against all the drug resistant isolates with MIC ranging between 0.01 – 1.41 mg/ml. The lowest MIC value of 0.01 mg/ml obtained in this study was shown by the acetone extract of S. lancea while the highest MIC value of 1.41 mg/ml was recorded with the acetone extract of T. emetica. Generally, the acetone extracts of all the plants showed better activity than their ethanol counterparts except for E. caffra. Ethanol is generally preferred as an extraction solvent in industrial applications, such as large-scale preparation of bioactive plant extracts, owing to its relative safety compared to acetone, which is more flammable. The S. aureus strains appeared to be more susceptible to the extracts than the NAS strains. An interesting finding was that the drug resistant isolates used in this study were generally more susceptible to the extracts than the ATCC strain which was in turn more susceptible to gentamicin, the positive control. All the plant extracts tested had LC50 values higher than the recommended cytotoxic cut-off concentration of 0.02 mg/ml. The ethanol extract of E. caffra had the best mean selectivity index (SI), calculated as LC50/MIC against all the pathogens, of 8.30. The antibiofilm investigation revealed that most of the plant extracts had very good inhibitory activity, inhibiting more than 50% of the test organism biofilm biomass, with E. caffra and A. venosum showing outstanding activities. The results also suggest that the S. epidermidis (NAS) ATCC strain was more susceptible to the antibiofilm activities of the plant extracts than the S. aureus isolate. The antioxidant activity investigation of the crude extracts of three of the plants showed that the ethanol extract of S. lancea had the best antioxidant activity against the 2, 2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals when compared to the other extracts. The ethanol extract of S. lancea had better anti-inflammatory activities than other extracts prepared using different solvents. The results reported in this dissertation demonstrate the potential of the selected South African plants to manage bovine mastitis as the plant extracts showed good antibacterial activities at relatively non-cytotoxic concentrations. The results further reveal the abilities of the plants to disrupt the biofilm-forming activities of drug-resistant Staphylococcus spp. implicated in bovine mastitis. The anti-inflammatory and antioxidant activities demonstrated by some of the plants also showed the potential of the plants to arrest oxidative stress and anti-inflammatory activities involved in the pathophysiology of the disease. S. lancea and E. caffra appeared to show great potential in the overall management of infectious bovine mastitis. Potential for synergistic antibacterial activity of the plant extracts needs to be examined for potential formulation of a polyherbal product. The active compounds need to be isolated and identified from the most active plant extracts to provide possible marker compounds for quality control if an active extract or fraction is to be developed for use against mastitis pathogens. The mechanism of action of the active plant preparations (extracts, fractions or isolated compounds) needs to be elucidated to identify how they can be most useful in preventing or treating diseases caused by mastitis pathogens for possible complementary applications in the management of bovine mastitis.
Endometrial glandular changes in aged captive cheetahs (Acinonyx jubatus) in southern Africa
(University of Pretoria, 2021-04) Hartman, Marthinus Jacobus; Clift, Sarah J.; lidavenant@gmail.com; Avenant, Alida
The cheetah, Acinonyx jubatus, is classified as “vulnerable” on the IUCN red list of threatened species. With declining population numbers in the wild, conservation efforts have focused on captive breeding and investigating the long-term effects of reproduction control strategies. Cheetahs in captivity survive longer than their wild counterparts and are purportedly more predisposed to conditions such as endometrial hyperplasia, cystic endometrial glandular changes, endometritis and pyometra. Uterine samples were collected from 21 captive aged cheetahs (mean and median age of 10 years) from three research conservation organisations in southern Africa. Endometrial changes from cheetahs sampled one year post-laparoscopic salpingectomy (n=7) were compared with samples collected at ovariectomy (n=7) or post mortem (n=7). Histological examination showed minimal endometrial pathology with only mild glandular ectasia (calculated as gland lumen area %) in most cases. This contrasts with previous studies in cheetahs, and other domestic and wild felids and canids where severe cystic and/or hyperplastic endometrial glandular changes were frequently reported. Increased age was positively correlated with increased gland lumen area (% and median) and endometrial area. There were no differences in endometrial changes between the post-salpingectomy and other samples. No notable endometrial changes were observed in the 10 cheetahs previously implanted with deslorelin as a contraceptive. It is possible that the observed mild endometrial changes are within the realm of physiological normality and not representative of overt pathology. This requires further investigation before new grading systems are developed to evaluate endometrial glandular pathology in cheetahs.
Optimal conditions for extraction of potentially therapeutic compounds from Pelargonium sidoides
(University of Pretoria, 2023-11-20) McGaw, Lyndy Joy; Famuyide, Ibukun Michael; joanvanwyngaard@gmaill.com; Van Wyngaard, Joan
Pelargonium sidoides (P. sidoides) DC., a member of the Geraniaceae family, is of significant importance in both traditional herbal practices and modern phytotherapy. It is widely used to treat bronchitis and infections of the upper respiratory tract. This herb has gained substantial recognition due to its scientifically established effectiveness and safety, as supported by clinical trials and previous patents. Its successful presence in the German and international markets underscores its popularity. The core of this herbal remedy is EPs® 7630, a unique aqueous-ethanolic (11 % (m/m) ethanol) root extract of P. sidoides. This extract is notably documented in the European Pharmacopoeia (EP). However, the existing pharmacopoeial method, which involves assessing tannin content in roots, is not ideal for evaluating final products that are ethanol extracts or dried root extracts. This limitation arises from the potential impact of the extraction solvent on various constituents. In particular, P. sidoides is reported to encompass a diverse array of compounds beyond tannins. Currently, the roots used for commercial purposes are sourced through wild harvesting in South Africa. This practice introduces significant variability, as existing studies have focused mainly on fluctuations in umckalin levels within the roots. Umckalin serves as a coumarin marker but is not necessarily reflective of the pharmacological effectivity (or bioactivity). Organisations that oversee wild harvesting only assess umckalin levels to gauge root quality. Despite extensive research, the precise active constituent within P. sidoides remains elusive. Interestingly, even today, only 80 % of the root composition has been characterised, leaving approximately 20 % unidentified. The Willmar Schwabe Pharma group of companies has conducted numerous clinical studies on Pelargonium, and particularly on their trademarked active ingredient EPs® 7630. As a result of these efforts, the European Medicines Agency (EMA) has endorsed an 11 % ethanolic extraction (1:8-10) and its equivalent dried root extract (4-25:1) to treat colds and flu. This has emerged as the industry standard, often referred to as the gold standard. Schwabe initially held a patent on the 11 % ethanol extraction; however, they withdrew it in 2010 to ensure fairness within the Pharma industry. A notable challenge arises from the absence of established quality control parameters to compare these roots extracts with each other or the market leader. Furthermore, the Schwabe product lacks standardisation to specific constituents, complicating matters even more. The specifications provided in the EP sole concern is the ethanol extraction percentage and the drug extraction ratio. A literature survey revealed that the choice of 11 % ethanol extraction lacks scientific justification. The approval from the EMA comes from the traditional use of the product and its market presence for more than three decades. Historically in South Africa, prior to the 2014 Complementary Medicine regulations, a common practice was to employ a 60 % ethanol extraction due to higher yields, reduced microbial contamination, and a streamlined process. With the regulatory shift, an exploration was initiated to assess the differences between various ethanol percentage extractions. Furthermore, an attempt was made to create an industry specification to help pharmacists evaluate P. sidoides root extract or products. With the introduction of the new regulations, it was decided to first evaluate what the difference would be between different percentage extractions of ethanol and then also to try and compile a standardised final product specification that can be used in the industry for pharmacists when evaluating the options of the root products of P. sidoides. Several critical issues have been identified within the context of P. sidoides products and their manufacturing. First, the practice of wild harvesting roots introduces significant variability due to the differing conditions, altitudes, and rainfall in various regions. This variability is further compounded by the maturity of the roots, which are typically harvested after four years. Second, there is a limitation in umckalin testing, as it primarily serves for species verification and does not provide insights into the root's activity. Third, leading trademarked extracts and dried root extracts lack standardisation with respect to specific constituents, relying mainly on the percentage of ethanol and the drug extraction ratio. This problem is exacerbated by the absence of specific regulatory guidance for P. sidoides products in South Africa, even after the introduction of complementary medicines guidelines by the South African Health Products Regulatory Authority (SAHPRA), forcing industry pharmacists to resort to European guidelines for guidance. Fourth, there is a notable absence of publicly available information concerning the distinctions between different percentages of ethanol used in extractions. Finally, quality control research has touched on various aspects, including distinguishing P. sidoides from related species and the significance of sulfated coumarins, especially in the context of COVID-19 research. In response to these challenges, there is an urgent need for a more robust approach to quality control and regulation that takes into account the diversity of products, regional variations, and the sustainable management of this valuable botanical resource. This thesis encompasses three distinct studies. The first study evaluated the impact of different concentrations of ethanol on the extraction of Pelargonium roots, focusing on yield, umckalin levels, and total polyphenols. The second study investigated the effects of 11 % and 60 % ethanol concentrations on the chemical fingerprint of the root extract and its bioactivity, including antimicrobial, anti-inflammatory, and immune modulating properties. This study also included an examination of the antiviral activity of a P. sidoides root extract against SARS-CoV-2. The final part of the thesis formulated a comprehensive quality control specification, integrating insights from previous research, with the goal of defining essential criteria for a dried P. sidoides root extract in line with the study findings for practical applications. The primary objective of this research was to systematically explore optimal sources and conditions for extracting potentially therapeutic compounds from P. sidoides, a vital medicinal plant indigenous to southern Africa, known for its efficacy against respiratory infections. This work aims to contribute to the development of formal specifications and criteria necessary for the registration of P. sidoides-based products in South Africa. The research in question was designed to achieve specific objectives aimed at advancing our understanding of P. sidoides and improving the quality control and regulation of its products. First, an exhaustive review of the existing knowledge surrounding P. sidoides was conducted, with a focus on its mechanisms of action, quality control methods, extraction techniques, chemical composition, in vitro activity, clinical applications, and considerations related to legal frameworks, procurement processes, and regulatory approvals. This served as a foundational knowledge base for subsequent investigations. Subsequently, representative root samples were obtained from two distinct geographic regions, the Eastern Cape and the Free State, where P. sidoides is traditionally sourced and harvested. These samples were crucial for subsequent analyses and comparisons. To further explore the chemical composition of P. sidoides, ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) technology was utilised to identify and quantify umckalin and umckalin sulfate in various batches of P. sidoides, with reference to a standard umckalin sample. The influence of various extraction methods was another key objective of investigation, with a specific focus on different concentrations of ethanol and extraction temperatures, as these parameters can significantly affect the chemical makeup of the root extracts. Using the same UPLC-QTOF-MS technology, a comprehensive chemical fingerprint unique to P. sidoides was established, adding to the knowledge of its constituents. To gauge the practical implications of these findings, the study delved into the biological activity of selected dried root extracts, comparing those extracted with 11 % and 60 % ethanol concentrations. The assessment included statistical analyses to identify differences between the chemical fingerprint and biological activity in various sample sets and to find potential correlations among the levels of selected constituents. Finally, one of the critical objectives was to develop quality control criteria that could be adopted by SAHPRA for the standardisation and regulation of P. sidoides root products. This step was designed to provide a framework to ensure the quality and consistency of P. sidoides-based products, contributing to the overall enhancement of their safety and efficacy within the market. The key findings and conclusions derived from this research include various aspects of the root extracts of P. sidoides. First, the high sensitivity of the UPLC-MS-QTOF method for quality control was demonstrated, with umckalin sulfate consistently identified as the predominant peak. Electrospray ionisation in negative mode proved to be more efficient than in positive mode, enhancing the method's reliability. Accurate umckalin quantification was found to be best achieved after hydrolysis, enabling precise values for stability studies and robust quality control. Distinctions were revealed in the roots of P. sidoides from different regions, with the Eastern Cape roots exhibiting higher umckalin levels but a lower polyphenol content, while those of the Free State showed the opposite trend. Despite relatively consistent coumarin levels in root samples, these differences were not statistically significant, necessitating further exploration with larger sample sets. Higher concentrations of ethanol and elevated extraction temperatures were highlighted to lead to increased levels of umckalin and polyphenols extracted from roots. The 30 % heated extraction method and the 80 % heated ethanol extraction resulted in the highest umckalin and total polyphenol levels. Furthermore, all the same peaks were identified in the 11 % and 60 % ethanol extracts, with only differing intensities. A comparison of the biological activity of root extracts with ethanol concentrations of 11 % and 60 % indicated advantages for the 60 % root extracts, including total minimum inhibitory concentration (MIC) activity, gram-positive and gram-negative activity, umckalin sulfate levels, and antifungal activity. Positive correlations were discovered between MIC values and total polyphenol levels, highlighting the significance of polyphenols in the root extract, which have associations with immunomodulation, anti-inflammatory effects, chemoprevention, neuroprotection, cardioprotection, and treatment for various diseases, as well as antibacterial and antiviral properties. In terms of specification for the dried root extract of P. sidoides, it was recommended to determine umckalin concentration after acid hydrolysis as a critical marker for differentiation between P. sidoides DC. and P. reniforme (Andrews) Curtis. Quantifying the total polyphenolic content was considered essential, with a minimum reference value of 15 % of total constituents. Additionally, methodically selecting the root harvest location could significantly enhance the potency of the root extract by increasing the proportion of polyphenolic compounds within the root extract. The hypothesis could also be proposed that umckalin is stored in the roots in a more water-soluble sulfate format, with evidence from acid hydrolysis indicating the detachment of the sulfate moiety from the umckalin molecule during the process. For precise umckalin quantification, umckalin levels were recommended to be quantified before and after acid hydrolysis. Lastly, identifying peaks and calculating total umckalin and polyphenol levels were advocated as a more accurate and comprehensive approach to quality control procedures, rather than relying solely on marker ratios. Additionally, a hypothesised synergistic interaction between coumarins and polyphenols suggested that polyphenols may enhance the bioavailability of coumarins, further emphasising the significance of considering the overall picture of both in quality control assessments of P. sidoides containing products.
Antimicrobial resistance in indicator bacterial species from wildlife at the human- livestock-wildlife interface in the Mnisi community, Mpumalanga, South Africa
(University of Pretoria, 2023-04) Naidoo, Vinny; Jonker, Annelize; amugochi@gmail.com; Mugochi, Lawrence
Antimicrobial resistance (AMR) is one of the greatest threats currently facing humanity. While there have been extensive studies on this subject in public health and livestock, there is paucity of information on the epidemiological role of wildlife in AMR. Anthropogenic activities have been suggested to be the main reason for presence of AMR in wildlife. Wildlife can also be a reservoir of naturally occurring resistance. While a one health approach has been put in place to tackle AMR, there have been obstacles in implementing it. The aim of this study was to determine the resistance of E. coli and Enterococcus isolates from wildlife faecal samples. Fifty-one wildlife faecal samples (herbivores and carnivores) collected from Mnisi between 2015 and 2020 and stored in a biobank at Hans Hoheisen Research Station were used in this study. Isolation of E. coli and Enterococcus was done on MacConkey and Blood agar and Biochemical tests done to identify isolates. Minimum inhibitory concentration was done using Thermo Scientific Sensititre plates according to manufacturer recommendations and interpreted using Clinical Laboratory Standards Institute standard tables. A total of 14 E. coli and 42 Enterococcus isolates were obtained. 52% of the Enterococcus isolates were identified as E. faecium and 48% as E. faecalis. Antimicrobial susceptibility analysis for E. coli showed one multidrug resistant isolate, 14% susceptibility to cefuroxime and 7% susceptibility to cefazolin, cefepime, ceftriaxone, cefoxitin and cefpodoxime and imipenem. Antimicrobial susceptibility for Enterococcus revealed 42% of isolates had multi drug resistance. Daptomycin, Rifampin and Quinuptistin/dalfopristin had highest resistance of 71%, 64% and 43% respectively. The proximity of the wildlife to livestock and human settlement is likely to be related to the high level of resistance as sewage wastes, run off water and environmental contamination may be responsible for the dissemination of resistant genes. The results of this study show that wildlife is a major player in antimicrobial resistance. The researchers recommend follow up studies and AMR surveillance in wildlife, human hospitals, livestock and water bodies in the area.

Browse

Recent Submissions