Master of Science / Department of Diagnostic Medicine/Pathobiology / Daniel U. Thomson / Escherichia coli O157:H7 is a human food-borne pathogen and cattle feces are a major source of contamination. Immunization against E. coli O157 may be a practical pre-harvest intervention strategy. A siderophore receptor/porin proteins (SRP) based vaccine has been developed to decrease the prevalence of E. coli O157 in cattle. Two studies were conducted to determine the efficacy of the SRP vaccine. In the first study, thirty calves were randomly assigned to one of two groups: control or SRP vaccine. Two weeks after the second vaccination, calves were orally inoculated with nalidixic acid-resistant (Nal[superscriptR]) E. coli O157. Fecal samples were collected for five weeks. Calves were necropsied on day 35 to collect gut contents and tissue swabs to determine Nal[superscriptR] E. coli O157:H7. The number of calves that were culture positive for E. coli O157 were lower (P= 0.07) in vaccinated group compared to the control. In the second study, cattle in two feedlots were randomized to SRP vaccine or control. Cattle were vaccinated on days 0 and 21. Rectal fecal samples were collected on day 0, and pen floor samples were collected on days 21, 35, and 70. Rectal fecal samples, RAMS, and hide swab samples were collected on d 85. Cattle were weighed on days 0, 21, and 85. Vaccination significantly reduced (P = 0.04) fecal E. coli O157 prevalence. There was also a decrease (P < 0.05) in E. coli O157 prevalence on hides and in fecal samples on day 85 in vaccinated cattle compared to the control.

Doctor of Philosophy / Department of Anatomy and Physiology / A. Philine Wangemann / Pendred syndrome is characterized by sensorineural deafness and post-pubertal goiter. It is caused by mutations in the anion exchanger, pendrin (SLC26A4). The purpose of this study was to understand the etiology of Pendred syndrome using a mouse model. Different methods of amplification from nanogram amounts of starting RNA were evaluated for gene array application. Gene arrays were performed and free radical stress markers were compared between the stria vascularis and the thyroid of the Slc26a4+/- and Slc26a4-/- mice. Hearing loss in Slc26a4-/- mice is linked to the loss of Kcnj10 protein expression and consequently the loss of endocochlear potential. To understand the mechanism of hearing loss in Slc26a4-/- mice, progressive loss of Kcnj10 protein expression in stria vascularis of Slc26a4-/- mice was assessed, the modulation of Kcnj10 protein expression by free radical stress in cultured stria vascularis and in an heterologous expression system was evaluated. To characterize the thyroid pathology, pendrin expression in the thyroid of Slc26a4+/- mice was measured in a developmental study and correlated with serum thyroxine (T4) levels of Slc26a4+/- and Slc26a4-/- mice over a developmental time course. All tested methods of RNA amplification were suitable for gene array application and demonstrated high internal consistency. Intermethod comparisons revealed variations in data, suggesting that a single amplification method ought to be used within a given experiment. Markers of free radical stress were increased in the stria vascularis of Slc26a4-/- mice before the onset of hearing. Progressive loss of Kcnj10 expression was seen in Slc26a4-/- mice at the onset of hearing. Furthermore, free radical stress modulated the expression of Kcnj10 in cultured stria vascularis and in a heterologous expression system. The pendrin mRNA expression was marginal in the thyroid and did not correlate with serum T4 levels. Further, absence of pendrin did not affect free radical stress markers in the thyroid. These data suggest that free radical stress-mediated loss of Kcnj10 expression in stria vascularis contributes to deafness in the Pendred syndrome mouse model and that pendrin is not essential for the function of mouse thyroid gland.

Master of Science / Department of Kinesiology / Timothy I. Musch / Advancing age alters the structural and functional determinants of convective and diffusive muscle oxygen (O[subscript]2) flux. However, capillary red blood cell (RBC) hemodynamics have not been investigated during contractions in muscles of old animals. PURPOSE: To test the hypothesis that aging induces significant alterations in capillary hemodynamics during electrically-induced contractions in the spinotrapezius muscle of old Fischer 344 x Brown Norway rats when compared to younger counterparts. METHODS: The spinotrapezius muscle was observed via intravital microscopy in 8 old (O: 26-30 months) and 5 young (Y: 6-8 months) animals. Wire electrodes elicited 1 Hz (6-8 volts) contractions for 3 minutes. RBC flux (FRBC), velocity (VRBC), capillary hematocrit (HCAP), and total microvascular O[subscript]2 delivery (QO[subscript]2m) were measured both at rest and during the steady-state of muscle contractions. RESULTS: At rest F[subscript]RBC and V[subscript]RBC were elevated in O compared to Y rats, while there was no difference in HCAP or QO[subscript]2m between groups. During the contracting steady-state, [Delta]F[subscript]RBC (Y: 28.8 [plus or minus] 7.7, O: -2.9 [plus or minus] 1.4 cells/s), [Delta]V[subscript]RBC (Y: 253 [plus or minus] 68, O: -4 [plus or minus] 15 [Mu]m/s), [Delta]H[subscript]CAP (Y: 0.02 [plus or minus] 0.02, O: -0.03 [plus or minus] 0.01 cells/[Mu]m), and [Delta]QO[subscript]2m (Y: 892 [plus or minus] 255, O: -24 [plus or minus] 30 cells/s/mm) cells/s/mm were all lower (P < 0.05) in O compared to Y rats. CONCLUSION: These results indicate that despite maintained total convective and diffusive O[subscript]2 transport at rest, advancing age results in significant alterations in capillary hemodynamics during electrically-induced contractions. These alterations likely contribute to the mechanisms responsible for the reduced exercise capacity commonly found in elderly populations.

The main aim of this study was to isolate and characterise antifungal and antibacterial compounds from leaf extracts of Combretum molle which belonging to the Combretaceae family. C. molle is one of the commonly used medicinal plants in southern Africa for numerous ailments. Three animal fungal pathogens, namely, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus and five plant fungal pathogens, namely, Aspergillus niger, Aspergillus parasiticus, Fusarium oxysporum, Penicillium janthinellum, Rhizoctonia solani and four nosocomial bacteria Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa were used as test microorganisms for bioactive compounds in leaf extracts of C.molle. Experiments for phytochemical analysis were done using different C. molle leaf extracts which were made using acetone, methanol, ethanol, ethyl acetate, chloroform, butanol and hexane as extractants. Thin Layer Chromatography (TLC) fingerprints of different leaf extracts were developed in three mobile phase systems, EMW, CEF and BEA and detected with vanillin-sulphuric acid spraying agent. The different extracts of C. molle showed the presence of many different compounds with distinct retardation factors (Rf), separated according to their polarities. Bioautography was carried out to determine the number of active compounds and their Rf values. The TLC plates were developed in three mobile systems, each sprayed with either fungal or bacterial strains. In BEA bioautograms of A. fumigatus, clear zones of inhibition were observed at Rf values of 0.12, 0.23, and 0.40. In EMW bioautogram of C. albicans, clear zones of inhibition were observed at Rf value of 0.73, 0.81, 0.87. C. neoformans had weak growth inhibition. Most of the fungal and bacterial strains tested in the bioautography displayed susceptibility to the active compounds, with P. janthinellum and P. aeruginosa showing exceptional sensitivity. The minimum inhibitory concentrations (MIC) values ranged from 0.02 to 2.5 mg/ml against the tested pathogens. The acetone and ethyl acetate extracts had the best inhibitory activity against P. janthinellum with an MIC value of 0.02 mg/ml. The acetone extract of C. molle gave the highest total activity (775 ml/g) against P. janthinellum. C. albicans was the most resistant pathogen with an average MIC value of 0.56 mg/ml compared with the other tested strains. Extracts were active against both Gram-positive and Gram-negative strains. P. aeruginosa extracts had the highest average MIC value (0.24 mg/ml) among the tested bacterial strains. In general, there was good overall inhibitory activity by different extracts of C. molle. Bioassay-guided fractionation of DCM extract of the leaves of C. molle yielded 32 fractions. Further fractionation led to the isolation of five compounds (C1, C2, C3, C4 and C5). Compound C1 was selected for structure elucidation due a larger quantity isolated and higher antimicrobial activity compared with the other isolated compounds. Nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy (MS) was used to show that compound C1 was taraxerol, belonging to the taraxerane group. Antimicrobial activity of the isolated compound against P. janthinellum had an MIC value of 0.08 ug/ml. Although the compound taraxerol have been discovered in other plant species, it is reported for the first time from C. molle in the study. The results illustrate that crude extracts and compound taraxerol from C. molle can be used as either an antibacterial or antifungal, and warrants further investigation. / Dissertation (MSc)--University of Pretoria, 2017. / Paraclinical Sciences / MSc / Unrestricted

Small stock farming and production accounted for approximately 8.4% of total animal product based agricultural output in the 2011 / 2012 season in South Africa. Large scale commercial farming aside, small stock farming also takes on an important role in poorer and developing rural areas of South Africa, where small stock are kept for a combination of economic and non-economic reasons including financial investment or security, food and resource production, as well as religious or traditional reasons. Blowflies are Dipterids with complex life cycles and complete metamorphoses, causing damage to hides and frequent death in their ovine hosts, as a result of cutaneous myiasis caused by the larval stages. All economically important blowfly species causing veterinary myiasis belong to the superfamily Oestroidea, which contains the three major families Oestridae, Calliphoridae and Sarcophidae. The two most significant blowfly genera in South Africa, Lucillia and Chrysomya, both belong to the family Calliphoridae. Chemical means of preventing and treating blowfly strike by topical application remains the most widely used method and appears to be indispensable at this stage. New molecules or formulations effective against blowfly strike are constantly being sought and form part of an active field of research. Bayer currently manufactures and markets Drastic Deadline Extreme ®, a pour-on formulation containing flumethrin and fluazuron for the control of blue ticks (Rhipicephalus decoloratus) in cattle; its possible action against blowflies in sheep was investigated in an in-vitro model, subsequent to a pilot pharmacokinetic study evaluating the kinetics of fluazuron when applied topically to sheep in this particular combination. The first objective of the project was to determine whether fluazuron has any effect at all on the development of blowfly larvae. An active ingredient from the same family of compounds, namely triflumeron, has been successfully used for several years to control blowfly strike in sheep in South Africa (Zapp ® Pour on - Bayer), but it was uncertain whether or not fluazuron would be effective. Raw fluazuron was applied to six pieces of beef according to a dose calculation based on the registered dose of the test product in cattle, while another six pieces were treated with saline in a similar fashion (n=6). Each piece of beef was placed in its own container along with six late instar larvae and placed in the incubator at 35°C for a further nine days. In this instance, the treated group demonstrated significant development defects with regard to pupation (uneclosed pupae) when analysed using the Mann-Whitney non-parametric t-test (p = 0.002). / Dissertation (MSc)--University of Pretoria, 2016. / Paraclinical Sciences / MSc / Unrestricted

Diatoms have a successful history of use in assessments of wetland biological condition. In North America and across Europe, diatom assemblages are used for routine wetland condition assessments to meet the statutory requirements of the European Water Framework Directive and the National Aquatic Resource Survey by the US Environmental Protection Agency. In South Africa, the use of diatom assemblages as indicators of wetland condition may be a promising alternative to the traditional biotic assemblages employed, such as macroinvertebrates or macrophytes, which have proven to be ineffective. We present a preliminary investigation on the feasibility of diatoms in wetland biological assessments in South Africa by evaluating the use of diatoms as indicators of biological condition for depressional wetlands in the Mpumalanga Highveld region of South Africa. Depressional wetlands typically found in this region are either temporary (seasonally inundated) or permanent depressions. Temporary depressional wetlands are expected to be affected by natural environmental disturbances (e.g., seasonal fluctuations in water-level which may cause changes in water chemistry) as compared to relatively stable permanent ones. Establishing whether diatoms are suitable indicators of natural environmental disturbances in temporary depressional wetlands in this region is necessary for further investigations of anthropogenic disturbances. We sampled epiphytic diatoms from three least human-disturbed temporary depressional wetlands during various stages of inundation and showed that the species composition of epiphytic diatom communities were strong indicators of temporally changing environmental conditions. Using the same diatom and physical and chemical data, we also demonstrated that simplifying the taxonomy by using the functional composition (ecological guilds, life-forms) of the epiphytic diatom communities, can assess temporally changing environmental conditions as effectively as the species composition. Moreover, these functional groups provide valuable ecological information that is not available from the species data. Acid mine drainage (AMD) is the predominant stressor in permanent depressional wetlands of the Mpumalanga Highveld region, where coal mines utilise these wetlands for storage of AMD, which has severe impacts on the structure and function of the ecosystem. In order to develop an approach for impact assessment and management of depressional wetlands in the region, we developed an epiphytic diatom multimetric index (MMI) for AMD impacted permanent depressional wetlands. This is also the first diatom index to quantify AMD impacts in wetland habitats. Data collected from 34 sites that represented a range of conditions along an AMD gradient within the Mpumalanga Highveld was used to select responsive diatom metrics which we combined into a multimetric index. We developed separate MMIs for classes of depressional wetland types in order to account for natural variation among diatom assemblages, and compared their performance with an MMI that did not account for natural variation. To account for natural variation, we classified reference sites based on diatom typologies and hypothesised that by using this approach, we would improve MMI performance. Overall, all MMIs performed considerably well, although grouping sites by diatom typology to account for natural variation improved MMI performance, especially the precision, responsiveness and sensitivity to disturbance. We conclude that diatoms have strong potential for use in wetland ecological assessments in South Africa. The experimental and statistical approaches used in this study should expand our knowledge of diatom ecology and further advance the research and development of diatom bioassessment. / Thesis (PhD)--University of Pretoria, 2017. / Paraclinical Sciences / PhD / Unrestricted

Sclerosteosis is a severe autosomal recessive sclerosing skeletal dysplasia with no available treatment. It is characterised by excessive bone formation and is caused by mutations in the SOST gene that lead to loss of expression of sclerostin, a protein that acts as a negative regulator of bone formation by binding to low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) Wnt co-receptors to inhibit the canonical Wnt/β-catenin signalling pathway. This study investigated the effectiveness of sclerostin replacement therapy in a mouse model of sclerosteosis. Recombinant wild type mouse sclerostin (mScl) and two novel mScl fusion proteins containing a C-terminal human immunoglobulin G1 (IgG1) antibody fraction crystallisable (mScl hFc), or C-terminal human Fc with a poly-aspartate motif (mScl hFc PD), to increase serum half-life and promote localisation to bone, respectively, were produced and purified using mammalian expression and standard chromatography techniques. These recombinant mScl proteins bound to LRP6 with high affinity (nM range) and completely inhibited matrix mineralisation in an in vitro bone nodule formation assay. Pharmacokinetic assessment following a single dose administered to wild type (WT) or SOST knock out (SOST-/-) mice indicated that the presence of the hFc increased protein half-life from less than 5 minutes to at least 1.5 days. The effect of a 6-week treatment with these proteins on the skeletal phenotype of young SOST-/- mice revealed that mScl hFc PD treatment resulted in a modest but significant reduction in trabecular bone volume compared with the vehicle control. There was no marked effect on cortical bone indices assessed by μCT, whole body areal bone mineral density by DXA, or terminal levels of the bone formation marker procollagen type 1 N-terminal propeptide (P1NP) in any of the SOST-/- or WT treatment groups, possibly due to insufficient exposure. Administration of recombinant mScl hFc PD protein partially corrected the high bone mass phenotype of the SOST-/- mouse, suggesting that bone-targeting of sclerostin engineered to improve half-life was able to negatively regulate bone formation in the SOST-/- mouse model of sclerosteosis. However, the modest efficacy indicates that sclerostin replacement may not be an optimal strategy to mitigate excessive bone formation in sclerosteosis, hence alternative approaches should be explored. / Thesis (PhD)--University of Pretoria, 2020. / UCB Pharma (Slough, UK) / National Research Foundation (NRF) / University of Pretoria (Pretoria, South Africa) / Paraclinical Sciences / PhD / Unrestricted

Mycoplasmas are a group of bacteria which are small in size, lack a cell wall, and have small genomes in comparison to other bacteria. The members of the group that are pathogenic utilize several mechanisms to evade the host's immune system. These processes affect surveillance and control mechanisms such as serologic testing and vaccination. Many of these organisms cause diseases of livestock, which heavily impact production parameters such as weight gain, milk yield, and egg production. Mycoplasmas also cause disease in people. Mycoplasma spp. can cause mastitis, metritis, pneumonia, and arthritis. The currently documented routes of transmission of Mycoplasma spp. are through fomites and by direct animal contact. The existence of environmental sources for Mycoplasma spp. and their role in transmission are poorly characterized. Mycoplasma spp. (confirmed as M. bovis using PCR) was found in recycled bedding sand from a dairy experiencing an outbreak of mycoplasma mastitis. The possibility of a persistent environmental source of Mycoplasma spp. in recycled bedding sand was further investigated using recycled sand from the dairy. Study objectives included determining factors associated with the persistence of Mycoplasma spp. in recycled bedding sand and the duration of survival of mycoplasmas in the sand. We also evaluated 2 disinfectants at 2 different concentrations each for the elimination of Mycoplasma spp. from contaminated sand. Mycoplasma spp. survived in the sand pile intermittently over a period of 8 months. The concentration of Mycoplasma spp. within the sand pile was directly related to temperature and precipitation. The survival of Mycoplasma spp. at a greater than expected range of temperatures suggests the formation of a biofilm. Ideal temperatures for replication of Mycoplasma spp. occurred between 15 °C and 20 °C. Moisture in the sand and movement of the sand pile also appeared to play a role in replication of mycoplasmas. Sodium hypochlorite (0.5%) and chlorhexidine (2%) were efficacious in eliminating Mycoplasma spp. from contaminated bedding sand. Recycled bedding sand could be an environmental source of Mycoplasma spp. infections, including M. bovis, in dairy cows. Future studies should investigate the contribution of this environmental source to the epidemiology of mycoplasma infections in dairy cattle and other ruminants.

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