Heartwater, caused by Ehrlichia ruminantium, causes significant economic losses to commercial small stock farmers, including commercial boer goat farmers in the Limpopo Province of South Africa. Heartwater is endemic in this region where Amblyomma hebraeum, the South African bont tick and the vector for this disease occurs. Due to their long mouthparts and specific attachment sites on goats, A. hebraeum is also associated with wounds, secondary abscess formation and lameness. Small stock farmers in the Limpopo Province employ various methods to control A. hebraeum and the negative effects associated with this tick, including heartwater. Two approaches where followed to focus on the control of A. hebraeum on the one hand and the detection and cryo-preservation of E. ruminantium from A. hebraeum on the other hand. The aim of this study was therefore twofold. By firstly determining the efficacy of a 1.0% fipronil pour-on solution against A. hebraeum by performing a therapeutic- and persistent efficacy trial and secondly to detect and cryo-preserve E. ruminantium from A. hebraeum ticks from a heartwater endemic region in the Limpopo province of South Africa. It was demonstrated that a 1.0% fipronil pour-on solution is 100% effective and has a persistent efficacy of seven days against A. hebraeum, but only if a targeted treatment approach is followed whereby the pour-on solution is applied to the predilection sites of A. hebraeum. A 1.0% fipronil pour-on solution can therefore be used by boer goat farmers to control A. hebraeum and the negative effects associated with this tick. In order to detect and cryo-preserve E. ruminantium from A. hebraeum ticks, ground-up-tick-supernatant samples were prepared in the laboratory from A. hebraeum ticks collected from goats and cattle at specified time intervals. To determine whether E. ruminantium was present in A. hebraeum ticks, DNA extraction, nested PCR of the pCS20 region of the E. ruminantium genome and demonstration of the amplified DNA by agarose gel electrophoresis was performed. The results demonstrated that E. ruminantium was indeed present in eight out of eight ground-up-tick-supernatant samples that were cryo-preserved. In future research, these samples may become very useful for isolating current strains of heartwater, which will ultimately lead to a better understanding of the disease and to facilitate the implementation of novel control methods. / Dissertation (MSc)--University of Pretoria, 2018. / Veterinary Tropical Diseases / MSc (Tropical Animal Health) / Unrestricted

Foot-and-Mouth Disease (FMD) is a disease of cloven-hoofed animals well-known not only for significant production losses but also restricted market access for livestock and livestock products from affected areas. It severely limits market opportunities for poor farmers and nations wishing to access more lucrative markets, both regionally and internationally. In the Northern Communal Area (NCA) of Namibia, FMD has not been detected in Kunene, Omusati, Oshana, Oshikoto and Ohangwena regions for the past 45 years or more, while outbreaks occurred in Eastern Kavango towards the end of 2008 as well as beginning of 2009 and there have been frequent outbreaks in the Zambezi region. Although, FMD has not been detected in five of the six regions of the Namibian NCA, no studies have been carried out to substantiate the FMD status in this highly potential livestock production area as required by the World Organisation for Animal Health (OIE). It is based on this fact that a study was carried out in the above mentioned regions to determine whether there is Foot-and-Mouth Disease Virus (FMDV) circulating in the cattle population of the NCA Namibia. A set of 4214 serum samples were taken from non-vaccinated animals against FMD in Area A, which included Kunene north, Omusati south, Oshikoto(NCA) and Oshana. Meanwhile, 5228 serum samples were taken from Area B, which consisted of Omusati north, Ohangwena and Kavango region where cattle had been vaccinated against FMD for the past 15 years or more. Samples were primarily screened for FMDV using the Ceditest® NSP ELISA and positive samples were subsequently tested with the Viral Neutralization Test (VNT). Herds containing animals that were positive on both tests were followed up and retested with the Ceditest® NSP ELISA and examined clinically for FMD signs. The initial results of the Ceditest® NSP ELISA in Area A and B revealed an apparent prevalence of 0.24% and 0.27% with corresponding 95% confidence intervals between 0.13, 0.44 and 0.16, 0.45 respectively. The follow up results found no clinical evidence of FMD and all subsequent Ceditest® NSP ELISA results were negative. The results indicate that the initial Ceditest® NSP ELISA positive cattle were false positive. Thus the overall outcome indicates that there is little chance of FMD virus circulation in the NCA Namibia. The survey also highlighted the need for taking into account sociological and cultural practices when designing a survey of communal cattle in order to get a representative sample of the cattle population. The study also recommended that Directorates of Veterinary Services (DVS) Namibia should maintain and continuously review FMD surveillance activities in the NCA, especially along the Angola – Namibia border and continuously harmonise these activities with Angolan counterparts. A more comprehensive study should be carried out to address the insufficient knowledge on sociological factors, which may influence sampling strategies in communal farming areas. / Dissertation (MSc)--University of Pretoria, 2015. / tm2015 / ab2016 / Production Animal Studies / MSc / Unrestricted

Protein is a fundamental part of nutrition in all animals but ruminants have the ability to metabolise and utilise non-protein nitrogen for this use. This allows for an economical alternative source of protein that does not compete with human resources. Previous evidence suggests that feeding high levels of protein to support increased demands for growth and production may have a negative impact on reproduction, since protein is rapidly converted to ammonia and further metabolised to urea which are both toxic to the reproductive system. Investigations within the South African climate indicated that the Nguni cow maintained higher blood urea nitrogen (BUN) levels during periods of drought than other cattle breeds. The Nguni breed is well adapted to extreme situations but as its popularity grows and the breed is promoted for emerging farmers it becomes essential to know if it possesses adaptations which could predispose it to reproductive failure if supplemented in the same manner as other commercial breeds. In this prospective experimental study, 22 multiparous cows of two breeds, the Hereford and the Nguni, were block randomised into a cross over design whereby all animals were fed a total mixed ration (TMR). The test group was provided with an increasing rumen degradable protein content in the form of feed grade urea. The control group was maintained on a urea free TMR. In the test group, the urea inclusion rate was increased weekly by 50 g to allow for rumen microflora adaptation and prevent acute urea toxicity. They reached a maximum inclusion rate of 200 g urea per cow per day. All animals had serum collected and oocytes aspirated by transvaginal ultrasound guided oocyte pick up (OPU) were counted and graded twice weekly. Following this, all usable oocytes were pooled per breed and treatment, and were subject to in vitro maturation, fertilisation and culture in order to assess the effect of elevated protein on oocyte competence and viability. Oocyte quality was defined as the number of oocytes with cumulus cells (Grade 1 3 oocytes) harvested per OPU, and oocyte viability was defined as the number of oocytes that cleaved by day 2 or that reached at least the morula stage by day 7 in the in vitro embryo system. Multivariable analyses were performed on BUN, antral follicle count (AFC) and number of grade 1 3 oocytes harvested per OPU session, and on the oocyte viability outcomes in the vitro embryo system. Nguni cows receiving 150 g dietary urea per day had lower mean BUN levels than Herefords (17.5 and 19.3 mg/dL respectively, P = 0.02). However dietary urea inclusion level, serum albumin level and sampling day were the only independent predictors of serum BUN. Nguni cows had lower mean antral follicle count (AFC) and number of oocytes harvested than Herefords (9.1 and 3.1, and 11.7 and 4.3 respectively, P < 0.01). The vet performing the aspiration, within day sampling order and BUN level >20 mg/dL were independently associated with the number of grade 1-3 oocytes after adjusting for AFC and the random effect of the individual animal. Increasing weighted mean serum albumin and decreasing weighted mean beta-hydroxybutyric acid (BHBA) were independently associated with the number of oocytes that cleaved (day 2) or that reached the morula stage (day 7) (P = 0.01 and P = 0.08 respectively). It was concluded that increasing dietary urea, low BCS and increasing serum albumin, but not breed, were associated with increasing BUN levels in cows. It was further concluded that BUN >20 mg/dL adversely affected the occurrence of cumulus cells around oocytes and that serum albumin and BHBA were, but neither dietary urea level nor breed was independently associated with viability of bovine oocytes in this study. Although the data were inconclusive about breed differences in protein metabolism, this study suggests that serum albumin has the potential to be used as predictor of the interaction between protein and energy metabolism and its effect on oocyte development. / Dissertation (MMedVet)--University of Pretoria, 2015. / tm2016 / Production Animal Studies / MMedVet

The South African crocodile industry has the potential to produce Nile crocodile skins, which are in high demand by the global fashion industry. The crocodile industry is an important economic resource to South Africa and Southern Africa. The main purpose of rearing Nile crocodiles at intensive production units is to produce viable hatchlings which will be grown to produce quality skins. The production of sufficient hatchlings depends on high hatching percentage of clutches. It has been observed that the hatching percentage varies among clutches. We do not know that whether the poor hatching percentages are associated with the microbial load from the incubation boxes or not. The aims of the study were to determine whether eggs in boxes that had more aerobic bacterial colonies and more fungal colonies before they received vermiculite and eggs are more prone to become lost from the pool potentially capable of yielding conceptuses developing to term than eggs in boxes that had fewer colonies before they received vermiculite and eggs, and whether there are species of aerobic bacteria, anaerobic bacteria and fungi that are present in unhatched eggs from clutches with low hatching percentage but not in clutches with high hatching percentage without the converse being true. At the time of hatching the farm personnel classify hatchlings or unhatched eggs into different categories e.g. hatchling survived, culled, foetus died in the shell, hatchling died, unfertilised egg, banded (the foetus died long before hatching), or rotten egg (putrefaction rendering the fertilisation status unknown). We assumed that banded or rotten eggs are more likely to have lost conceptuses due to the microbial load that was present in the incubation boxes before they were loaded with vermiculite and eggs than the other classes of eggs. A response variable Perhapsloststatus was therefore created. For each egg that was either classified as Banded or Rotten Perhapsloststatus was assigned the value of one. For each egg belonging to other categories Perhapsloststatus was assigned the value of zero. Eggs classified as unfertilised were excluded from Perhapsloststatus. The microbial load of aerobic bacteria and fungi from the incubation boxes before they were loaded with vermiculite and eggs was measured as colony forming units (CFU). The independent variable of interest was Count category (a categorical variable of aerobic bacteria and fungi colony counts), which was compiled as follows: 0 = Counts zero to below the 25th percentile, 1 = Counts from the 25th percentile to below the 50th percentile, 2 = Counts ranging the 50th percentile to below the 75th percentile, 3 = Counts from the 75th percentile to the maximum that was countable and 4 = Too numerous to count. Sterile swabs were used to collect samples from unhatched eggs for isolation and identification of aerobic bacteria, anaerobic bacteria and fungi from the 10 boxes with the lowest and highest hatching percentages, respectively. We used a population-averaged mixed-effect logistic regression model to determine the effect of Count category on Perhapsloststatus. The odds for eggs being banded or rotten (Perhapsloststatus = 1) in boxes with too numerous to count aerobic bacterial colonies (Count category 4) tended to be higher than for eggs in boxes with colony counts between zero and the 25% percentile (Count category 0) (P = 0.06). The odds of eggs being banded or rotten (Perhapsloststatus = 1) in boxes with fungal colony counts falling in any count categories 1 to 4 were the same as the odds of eggs being banded or rotten in boxes with fungal colony counts falling in Count category 0. The variety and frequency of species of aerobic bacteria, anaerobic bacteria and fungi from unhatched eggs of lowest hatching boxes and highest hatching boxes was almost the same. The species isolated from unhatched eggs are more likely pertaining to contamination from the cloaca of female crocodile or from the environment i.e. nesting material, personnel handling, vermiculite, incubation box or the incubator environment, as most of the isolated species present are normally present in soil, water, dust and also opportunistic pathogens in animals. This observational study shows that there is a trend of association between the aerobic bacterial load from the incubation boxes and the eggs in them to become either banded or rotten. / Dissertation (MSc)--University of Pretoria, 2015. / tm2016 / Production Animal Studies / MSc

Giraffes (Giraffa camelopardalis) have evolved into a unique and extreme shape. The principle determinant of its shape is the skeleton and the overarching theme of the study was to describe how this shape is achieved throughout ontogeny. Accordingly, the study had three main objectives: 1) To describe the growth of the giraffe postcranial skeleton allometrically, 2) To interpret the allometric patterns described in an evolutionary and functional sense and 3) To reconstruct the size and shape of the extinct Giraffa sivalensis using, if feasible, allometric equations obtained in this study. Secondary objectives were to a) establish if sexual dimorphism was evident in G. camelopardalis and b) determine if growth patterns in the foetus differed from those in postnatal G. camelopardalis. Data were collected from giraffes culled as part of conservancy management in Zimbabwe. The sample included 59 animals from which vertebral dimensions were taken in 48 animals and long bone dimensions in 47 animals. Body masses ranged from 21 kg to 77 kg in foetuses and 147 kg to 1412 kg postnatally, representing 29 males and 30 females. In addition to body mass, external body dimensions were recorded from each animal. Each vertebra and unilateral long bone was dissected from the carcasses and cleaned, after which dimensions were measured with a vernier calliper, measuring board or measuring tape. Vertebral dimensions measured included body (centrum) length, height and width as well as vertebral spinous process length. Long bone dimensions included length, two midshaft diameters and circumference. Allometric equations (y=bxk) were constructed from the data, with special interest in the scaling exponent (k) to illustrate regions of positively allometric, isometric or negatively allometric growth. In the first series of analyses the growth patterns of the components of the postcranial axial skeleton were analysed. The adaptations in vertebral growth to create and maintain extraordinary shape were identified as disproportionate elongation of the cervical vertebrae after birth, increasing cross sectional diameters of the cervical vertebrae from cranial to caudal and positively allometric spinal process growth. The theory of sexual selection as a driver for neck elongation in giraffes was brought into question by showing that male and female vertebral elongation rates are similar relative to increases in body mass. The second series of analyses described the growth pattern of the long bones of the appendicular skeleton. The allometric exponents seemed unremarkable compared to the few species described previously, and it was shown that the giraffe appendicular skeleton does not elongate in the dramatic way the neck does. Limbs at birth, after lengthening with positive allometry in utero, are already elongated and slender in shape and a further increase in the gracility of the bones is either not possible or not desirable. This result implies that it is neck elongation rather than leg elongation that is the dominant factor in the evolution of the giraffe shape. Nevertheless, the front limb bones and especially the humerus may show responsiveness to increasing high loads and/ or bending moments, which may be caused by the neck mass which increases with positive allometry, or with behaviours such as splaying the forelegs during drinking. In the third component of the study ontogenetic allometric equations in extant giraffes were applied to the remains of an extinct giraffid, G. sivalensis. The procedure was unusual as it employed ontogenetic regressions instead of the more commonly used interspecific regressions. The appropriateness of each equation to estimate body mass was evaluated by calculating the prediction error incurred in both extant giraffes and okapis (Okapia johnstoni). It was concluded that, due to body shape, ontogenetic equations were adequate and perhaps preferable to interspecific equations to estimate proportions in Giraffa species. This analysis showed that G sivalensis was smaller than extant giraffes and weighed around 400 kg (range 228 kg 575 kg), with a neck length of about 147 cm and a height of 390 cm. There may be evidence of sexual dimorphism in this species, with males being about twice the body weight of females. However, if sexual dimorphism was not present and all the bones were correctly attributed to this species, then G. sivalensis had a slender neck with a relatively stocky body. In conclusion, this study established ontogenetic regression equations for the skeleton of an animal of which the body shape seems to be at the extreme limits of mammalian possibility. The value of the current study lies especially in its sample size and quality, which included an unprecedented number of giraffe body masses, vertebral and long bone dimensions. This dataset had applications in the giraffe s evolutionary biology, palaeontology and even ecology. Future studies still need to compare the findings from giraffe growth with similar data from other taxa, especially those with long legs and necks. Specifically, it would interesting to determine if positively allometric neck growth combined with isometric leg growth is found in other mammalian species. In addition, the strength of giraffe long bones and vertebrae needs to be investigated with more accuracy using parameters like second moment of area. Lastly, further palaeontological studies on other giraffid sizes are necessary to validate the current and future interpretations of fossil giraffid findings. / Thesis (PhD)--University of Pretoria, 2015. / tm2016 / Production Animal Studies / PhD

Like many other wildlife species, blue wildebeest (Connochaetes taurinus) are routinely captured for relocation purposes. Several studies have shown that physiologically this is a very stressful period for wild animals and can affect several aspects of their well-being. Little is known about the level of physiological stress experienced by blue wildebeest during capture and confinement before transport. A non-invasive approach to assess adrenocortical function as a measure of physiological stress would be preferable, as animals are not disturbed during sample collection, and therefore sampling is feedback free. Currently, however, such a non-invasive measure has not been evaluated for any wildebeest species. An adrenocorticotropic hormone (ACTH) challenge test was performed on one sub-adult male and one adult female. We examined the suitability of five enzyme immunoassays (EIAs), detecting faecal glucocorticoid metabolites (fGCM) with a 5?-3?-ol-11-one (3?,11oxo-CM), 5?-pregnane-3?,11?,21-triol-20-one (37e), 11?,21-diol-20-one (corticosterone), 11,17,21-trihydroxypregn-4-ene-3,20-dione (cortisol), and 11,17-dioxoandrostane (11,17-DOA) structure respectively, for monitoring stress-related physiological responses in blue wildebeest. After evaluating all five EIAs, it was concluded that the EIA measuring 11,17-DOA performed the best for detection of fGCMs in blue wildebeest with a 21 fold increase above the baseline in the male and a 4,7 fold increase above the baseline in the female at 12 hours post ACTH injection. Under field conditions the use of the 11,17-DOA EIA allowed the monitoring of fGCM alterations when wildebeest were captured using a mass capture technique; fGCM concentrations were elevated by 1,5 times in samples collected from animals restrained for up to 10 hours in a truck compared to those collected directly after capture (P <0,001). No significant increase in fGCM concentrations was detected in faecal samples collected directly after capture from animals of the same herd captured either on day 1 or day 2 of mass capture, indicating that the presence of a helicopter during the two days of capture was not associated with a profound stress response in wildebeest that were not captured at the time but were in the vicinity of the capture. Storage of faeces in labelled plastic containers at ambient temperature for up to 48 hours post-defecation resulted in a significant decrease in 11,17-DOA levels from 8 to 48 hours after defecation. Therefore sample collection for 11,17-DOA determination in wildebeest can occur for up to 8 hours post-defecation without the risk of substantial decreases in 11,17-DOA concentrations. By identifying a suitable assay for determining 11,17-DOA concentrations in blue wildebeest, it can now be used by researchers, wildlife managers and veterinarians to reliably monitoring the physiological stress responses to capture or other management procedures with minimum interference to the animals. We have also shown that the presence of a helicopter during capture does not appear to have a lasting influence on wildebeest 11,17-DOA concentrations, but prolonged restraint for 10 hours does. / Dissertation (MSc)--University of Pretoria, 2016. / Paraclinical Sciences / MSc / Unrestricted

An unacceptable number of wild animals die or experience morbidity as a result of capture-related complications. Capture-induced hyperthermia is believed to play a role in the morbidity and mortality of captured animals. The aims of this research were to gain a better understanding of the pathophysiological consequences of capture and capture-induced hyperthermia and to better understand the association between capture-induced hyperthermia and capture myopathy. We aimed to determine whether cooling could reduce the pathophysiological consequences of capture and protect against capture related complications like capture myopathy. Forty wild blesbok were captured from the wild and then housed in bomas at Groenkloof Nature Reserve for the duration of the study. These animals were divided into three groups which included a group of animals that were chased and cooled during immobilization (C+C), a group that was chased but not cooled during immobilization (CNC) and a control group that was not chased before or cooled during immobilization (Ct). The control group received powder diazepam in their feed two hours before they were darted to minimize their stress response to capture. The treatment groups were chased for 15 minutes before they were darted. All the animals were darted and immobilized with etorphine and azaperone. The chased and cooled group were doused with 10L of 4°C water over a 10 minute period during the immobilization. The immobilization period lasted for forty minutes in all the animals. A number of clinical and physiological parameters were measured in all the animals directly after induction into immobilization, 40 minutes later, and then on day 2, 16 and 30 post the initial capture. On these subsequent days the animals were not chased but they were simply all immobilized in their holding bomas, for data and sample collection, after they received in feed diazepam. The parameters measured and analysed included muscle and rectal temperature, PaO2, PaCo2, pH, lactate, GGT, GLDH, creatinine, BUN, CK, AST, cardiac troponin I and cortisol. The animals in the control group had normal values for all the variables measured except they were hypoxic during the immobilization. The animals that were chased developed hyperthermia, hypoxia and mild acidaemia, and they had mildly elevated concentrations of GLDH, AST, CK and creatinine indicating mild hepatic, renal and skeletal muscle damage. These animals also had severe increases in lactate and cardiac Troponin I concentrations indicating cardiac damage possibly as a result of the hypoxia that occurred during the chase and the immobilization. Despite cooling correcting the hyperthermia, it did not prevent or protect against any of these pathophysiological effects. Therefore, this capture-induced hyperthermia appeared to play a limited role in causing these effects. Therefore, capture that involves chasing animals before immobilization appears to primarily cause cardiac muscle damage. This capture-induce cardiomyopathy may have profound secondary consequences, which could result in mortality or other capture related complications. / Dissertation (MSc)--University of Pretoria, 2016. / Paraclinical Sciences / MSc / Unrestricted

Since 1982 farmers and veterinarians mainly from the North West province of South Africa noticed an increase in cattle with associated lameness and osteopathy of unknown aetiology. Affected cattle presented with varying degrees of lameness as well as peri-articular swelling especially of the stifle joint. Cattle of all ages, particularly those between the ages of 6 - 18 months developed lesions. Lesions were also noticed among different breeds, production stages and different farming enterprises (commercial and stud farming). The only common factor among the affected cattle was the environment in which they were farmed. Culling of affected animals, especially calves, reduced the number of replacement stock to a level where some farmers were not able to produce sufficient replacement stock, consequently suffering tremendous financial losses. Investigation into the aetiology of the new sporadic osteopathy was deemed necessary. Initially it was suspected that the condition was due to mineral deficiency, as affected cattle fed a supplement containing high levels of micro- and macro minerals responded positively within three weeks during a pre-trial. The study was conducted to determine the level of micro- and macro minerals required to be included in a supplement that would improve the clinical condition of cattle clinically affected by osteochondrosis. Results indicated no significant difference between supplemetary treatments. A comparison between the pre-trial and the current supplements indicated that the supplements in the present study had negative dietary cation anion difference (DCAD) values. Since negative DCAD compositions may result in mild metabolic acidosis, as supported by literature, and the acidic urine collected in a small sample of the test cattle, the hypothesis is advanced that it is unlikely that supplementation, regardless of its mineral concentration will improve the condition of the cattle if the DCAD value of the supplement is strongly negative. The hypothesis should be tested in forthcoming research and the following should be determined: (1) the specific effect a supplement with a negative DCAD value has when fed to cattle with clinical osteochondrosis, and (2) the optimal DCAD value of a supplement that would aid in limiting the severity of osteochondrosis in cattle. / Dissertation (MSc)--University of Pretoria, 2016. / Paraclinical Sciences / MSc / Unrestricted

Farmers in developing countries are faced with many diseases that limit the productivity of their animals, several of which are caused by tick infestations. To date, treatment of host animals with synthetic, chemical tick repellents and acaricides remains the method used to reduce the influence of the parasites on animal and human health. Awareness of the environmental health hazards posed by these acaricides, development of tick resistance leading to recurrent ectoparasitism, danger of misuse and presence of toxic residues in food, water and animal by-products has led to the search for safe and environmentally-friendly alternatives, one of which is the use of medicinal plants. Because there appears to be a need and to contribute to research in this field, extensive literature surveys of published scientific articles were conducted. The following aspects were addressed: the role of ticks in animal health, problems encountered in using synthetic, chemical acaricides, medicinal plants with in vitro acaricidal or tick repellent activities against immature and adult stages of ticks and bioassays employed. Veterinary databases (All Databases, CAB Abstracts and Global Health, Medline, Pubmed, Web of Science, BIOSIS Citation Index, Science Direct, Current Content Connect and Google Scholar) were searched. The search words included “acaricidal”, “tick repellent”, “medicinal plants”, “isolated compounds” and “antitick assays”. Meta-analysis was conducted using the Fixed-effect model in an Excel programme to compare the results. The tick climbing repellency and adult immersion tests were the most commonly used assays to test for repellency and acaricidal activity respectively. Ethanol was the most commonly used extractant and Rhipicephalus (Boophilus) microplus was the most commonly studied tick across all the reviewed papers. More than 200 plant species from several countries globally have tick repellent or acaricidal properties discovered using in vitro assays. A large proportion of the published work was done in tropical countries such as Brazil, India and South Africa where ticks cause major problems. The median efficiency values for acaricidal, larvicidal, egg hatching inhibition, inhibition of oviposition, repellency, acaricidal effects of the Lamiaceae and Asteraceae family using a total of 1428, 1924, 574, 281 and 68 events were 80.12 (CI95%: 79.20 - 81.04), 86.05 (CI95%: 85.13 - 86.97), 83.39 (CI95%: 82.47 - 84.31), 53.01 (CI95%: 52.08 - 53.93), 92.00 (CI95%: 91.08 - 92.93), 80.79 (CI95%: 79.87 - 81.71) and 48.34% (CI95%: 47.42 - 49.26) respectively. Extracts of some species including Azadirachta indica, Gynandropsis gynandra, Lavandula angustifolia, Pelargonium roseum and Cymbopogon species have good acaricidal and larvicidal activities with 90-100% efficacy, comparable to those of currently used acaricides, although, usually at higher dosages. Compounds with acaricidal activity such as azadirachtin, carvacrol, linalool, geraniol and citronellal were listed. As a country, South Africa is rich in vascular plant flora, possessing over 10% of the world’s vascular floral species. Only a fraction of its plants have been rigorously studied and analyzed for their biological activity against ticks and seventeen plant species based on their ethnoveterinary use in tick control were selected for this study. The plants are Aloe rupestris Baker, Antizoma angustifolia (Burch.) Miers ex Harv., Calpurnia aurea subsp. aurea (Aiton) Benth., Cissus quadrangularis L., Clematis brachiata Thunb., Cleome gynandra L., Ficus sycomorus L., Gnidia deserticola Gilg., Hypoxis rigidula Baker var. rigidula, Maerua angolensis DC., Monsonia angustifolia E. Mey. ex A.Rich., Pelargonium luridum (Andrews) Sweet, Ptaeroxylon obliquum (Thunb.) Radlk, Schkuhria pinnata (Lam.) Kuntze ex Thell., Sclerocarya birrea (A.Rich.) Hochst., Senna italica subsp. arachoides (Burch.) Lock. and Tabernaemontana elegans Stapf. Crude extracts of the above mentioned plants were prepared using four different solvents (acetone, ethanol, ethanol/water and hot water). The extracts at a concentration of 200 mg/ml were screened for their acaricidal efficacy against adult Rhipicephalus turanicus ticks using the contact assay. The plant species with the highest acaricidal efficacies for their acetone and ethanol extracts were C. aurea, S. pinnata and S. italica with mortality of 97, 93, 90% and 93, 93, 87% respectively. The ethanol/water and hot water extracts of many of the plants had low acaricidal activities (<60%). An acaricidal dose-response bioassay of two-fold graded decreasing concentrations (100 to 3 mg/ml) of the acetone and ethanol extracts of S. pinnata, C. aurea and S. italica was determined using the adult immersion tests. The LC50 values for the acetone extracts were 35.75, 111.24 and 42.05 mg/ml respectively and for the ethanol extracts were 37.07, 98.69 and 37.50 mg/ml respectively compared with the positive control (cypermethrin) with LC50 of 2.41 mg/ml. In order to evaluate the potential safety of these plants, cytotoxicity against Vero and HepG2 cells was determined. Most of the plant extracts were non-cytotoxic to the two cell lines (LC50>100 μg/ml) and there was a statistically significant higher toxicity to HepG2 cells compared with Vero cells. The ethanol/water and hot water extracts of most of the plants were less toxic to the cells (LC50>1000 μg/ml) compared with their acetone and ethanol extracts. The selectivity indices of S. pinnata, C. aurea and S. italica were low. Particularly good acaricidal activities were displayed by C. aurea subsp. aurea extracted using four different solvents on R. turanicus ticks. The plant extract also had lower cytotoxicity against the cell lines tested and was selected as the most promising plant species, based on its efficacy and potential safety for further studies. / Thesis (PhD)--University of Pretoria, 2017. / Paraclinical Sciences / PhD / Unrestricted

The aim of this study was to investigate if a vaccine can be developed against epoxyscillirosidine, to prevent yellow tulp poisoning in livestock. Moraea pallida Bak. (yellow tulp) poisoning is the most important cardiac glycoside toxicosis in ruminants in South Africa. Cardiac glycoside poisonings collectively account for about 33 and 10% mortalities due to plants, in large and small ruminants, respectively. The toxic principle contained by yellow tulp 1α, 2α-epoxyscillirosidine, is a bufadienolide. Epoxyscillirosidine, proscillaridin and bufalin, were conjugated to [hen ovalbumin (OVA), bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH)]. Adult male New Zealand White rabbits were vaccinated in 3 trials. In trial 1 (T1) and 2 (T2), experimental (n=7) and control (n=5) animals were vaccinated with epoxyscillirosidine-OVA (0.4 mg/rabbit) and OVA (0.8 mg/rabbit), respectively. In T1 Freund’s (complete and incomplete) and in T2 Montanide was used as adjuvant, respectively. In Trial 3 (T3), five equal groups of 3 animals each, were vaccinated with proscillaridin-BSA (group 1), bufalin-BSA (group 2), epoxyscillirosidine-KLH (group 3), epoxyscillirosidine-BSA (group 4) and BSA (group 5), with Montanide as adjuvant, on days (D) 0, 21 and 42 (0.8 mg/rabbit, intradermally). Blood was collected before each vaccination and at 3 weeks after the last vaccination. Antibody response was determined using an indirect ELISA. There was a poor immune response associated with the dose and/or adjuvant in T1. However, after increasing the dose of the immunogen to 0.8 mg (per rabbit) and changing the adjuvant to Montanide, in T2 and T3, antibodies against the conjugates were successfully raised. In T3, epoxyscillirosidine-KLH (group 3) induced the highest immune response. Furthermore, proscillaridin and bufalin antibodies cross-reacted with epoxyscillirosidine and its OVA conjugate in the ELISAs. Preparatory to in vitro studies to assess the efficacy of the raised antibodies to neutralize epoxyscillirosidine, a rat embryonic cardiomyocyte (H9c2) cell line was established and the cytotoxic effect of epoxyscillirosidine was determined. Cells (10 000/well) exposed to epoxyscillirosidine (10–200 μM) for 24, 48 and 72 h were evaluated using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the lactate dehydrogenase (LDH) assays. Cells (100 000/well) exposed to epoxyscillirosidine (40–200 μM, for 24, 48 and 72 h), were processed and viewed with a transmission electron microscope (TEM). Cell viability indicated a hormetic dose/concentration response, characterized by higher viability (relative to control) at low doses (10–40, 10 and 10–20 μM for 24, 48 and 72 h, respectively) and decreased viability at higher doses. The cytotoxic effect and ultra-structural changes were dose and time dependent. Numerous cytoplasmic vacuoles, karyolysis and damage to the cell membrane, indicative of necrosis, were observed. The animal vaccination trial was scaled up, to generate more antibodies, for the in vitro neutralization studies. Six, adult Mutton Merino whethers were vaccinated with epoxyscillirosidine-KLH (2 mg subcutaneously), on D0, 21 and 42. Immune response was determined with an indirect ELISA. Antibodies were concentrated and purified using ammonium sulphate precipitation, before evaluation of in vitro neutralization efficacy. There was no significant (p > 0.05) difference in viability, between cells exposed to a pre-incubated solution of antibodies and epoxyscillirosidine and the epoxyscillirosidine exposed control cells. The antibodies failed to neutralize the toxic effect of epoxyscillirosidine. In conclusion, conjugated epoxyscillirosidine was an effective immunogen following conjugation to carrier proteins and antibodies were raised in vaccinated animals. Although antibodies against epoxyscillirosidine-KLH were raised in sheep, they failed to neutralize the toxin in the in vitro H9c2 cell model. This is possibly because higher ratios of antibodies to toxin are needed to effectively neutralize epoxyscillirosidine than those used in this study. Since antibodies failed to neutralize epoxyscillirosidine in the current study, further studies could optimize the vaccine to produce more specific antibodies with stronger affinity and avidity to be able to neutralize epoxyscillirosidine. Furthermore, the antibody purification method could be adjusted or changed for optimal results in the future. Antibodies against the related commercially available bufadienolides, namely proscillaridin and bufalin, cross-reacted with epoxyscillirosidine and could be investigated in future studies to prevent yellow tulp poisoning by vaccination. / Thesis (PhD)--University of Pretoria, 2018. / Paraclinical Sciences / PhD / Unrestricted